Transposable elements
any DNA sequence capable of moving from one place to another within the genome, it plays an important role in shaping the structure of chromosomes and genomes, which often cause chromosome duplications, deletions, and other types of rearrangements.
DNA has 3 hierarchy levels
primary - structure of DNA is it's nucleotide sequence
Secondary - the double stranded helix
tertiary - higher-order folding that allows DNA to be packed into the confined space
One type of DNA tertiary structure is, which is how chromosomal DNA is tightly packed to fit into the small confines of a cell
supercoling
When does Supercoiling take place
when DNA helix is subjected to strain by being overwound or underwound, when the strain of overrotating and underrotating cannot be compensated by the turning of the ends of the double helix, which is the case if the DNA is circular, that is there are no
Molecules that are overrotated
positive supercoiling, helix supercoliis in the same direction of the helix, right handed.
molecules that are under-rotated
negative supercoiling, helix supercoils in the opposite direction of the helix, left handed. DNA itself is always negative supercoiling.
supercoling relies on topoisomerases which are?
enzymes that add or remove rotations from the DNA helix by temporarily breaking the nucleotide strands, rotating the ends around each other, and then rejoining the broken ends, thus topoisomerases can both induce and relieve supercoling.
which statement is true regarding negative supercoiled DNA?
negative supercoiled DNA is under-rotated and allows for easier strand separation during replication and transcription.
2 advantages of DNA being negatively supercoiled
1) - makes the separation of 2 strands of DNA easier during replication and transcription
2) separation of the 2 strands during replication and transcription is more rapid and requires less energy
-also supercoild DNA can be packed into a smaller space th
A circular DNA molecule 300 bp long has 20 complete rotations. This DNA molecule is
negatively supercoiled.
300/10 - 30 rotations
only 20 rotations means its under-rotated
what if the circle is broken?
if the sugar phosphate backbone is broken, the torsional stress can be relieved by the helix rotating around the central axis
molecules will spotaneously go to a fully relaxed state
linear dsDNA does not have supercoiling unless one end is constrained.
enzyme that add or remove rotations from a DNA helix
topoisomerases
cleave the phosphodiester bond of the sugar-phosphate backbone
topoisomerases
Type 1 Topoisomerases does
cleaves only one strand of the double stranded DNA, includes Topo1A and Topo1B
Topo1A
prokaryotic enzyme that only relaxes negative supercoils
Topo1B
Eukaryotic enzyme can relax DNA or add positive supercoils
Type II Topisomerases
1)-breaks both strands and can add or remove rotations, depending on the specific enzymes
2)-structurally and bicohemically distinct from type 1
3)-more varied and complex functionality than type 1
bacterial chromosome facts
1) circular genome, not a giant relaxed circle
2) their DNA is associated with a limited amount of protein, far less than found in eukaryotic chromatin
3) located as a clump in distinct part of the cell called nuceloid.
Eukaryotic chromosome
1) interphase chromatin is relatively uncondensed (looses configuration, can't distinguish chromosomes)
2) it is less condensed than metaphase chromosomes
3) yet interphase chromatin is still highly condensed and very structured
two basic types of chromatin
euchromatin, heterochromatin
euchromatin
undergoes condensation and decondensation throughout the cell cycle, contains most the transcribed DNA, DNA which is used for synthesis of RNA
Heteorchromatin
1) remains highly condensed during the cell cycle, found at the centromeres and telomeres of all chromosomes, found also at other specific sites along chromosomes.
2) entire inactive x chromosome in female mammalian cells
3) until recently, thought there
Histones
1) basic proteins
2) major proteins associated with chromatin, they are positively charged at physiological ph
3) high percentage of arginine and lysine
4) attracted to the negative charge of DNA
histones are some of the
most highly conserved proteins known
five major histones
H1, H2A, H2B, H3 and H4, account for 50% of the protein associated with chromatin
Nonhistone proteins
1) the other 50% of chromsomal proteins
2) very heterogeneous collection
3) some nuclear proteins are not associated with chromatin
4) not always easy to determine
Nucelosome are
1)isolated chromatin, which looks like beads on a string when viewed with electron microscope
2) if you add a small amount of DNAse, the string is nicked
3) individuals beads with about 200 bp DNA
histone genes only expressed in
S phase or made only during S phase
Nuclease Protection Assay
protein protects the DNA from degradation or nuclease.
experiments show that chromatin has
a fundamental repetitive structure
chromatosome
1) core particle is 8 proteins, two copies each of H2A, H2B, H3 and H4.
2) histones H1 is not part of the core particles
3) H1 binds to the DNA at the site where the DNA exists the nucelosome
4) helps bind the DNA on the surface of the nucleosome octamer
H1 associated with
20-22 bp segment of DNA
chromatosome has
167 bp by DNA
145 - 147 bp on the nucleosome core and 20 bp by associated with H1. Each chromatosome is connected by -30-40 bp of linker DNA.
Transposable Elements
also called transposons, mobile elements, jumping genes and controlling genes
Transposable elements are first identified in
maize by Barbara McClintok in the late 1940's
Many eukaryotic genomes contains
significant amounts of DNA of DNA related to these transposons.
nucleosomes coil to form
a 30 nm fiber called solenoid
30 nm fibers form
large loops that are attached at their base to the chromosome scaffold, each loop is 20,000-100,00 bp DNA
Adjacent Nucleosomes Form
a 30 nm fiber Solenoid
Solenoid
these loops are the basic structure of interphase chromatin
chromatin condenses further to a 250nm fiber and finally a 700nm wide fiber to form the metaphase chromosome.
polytene Chromosomes
1)giant chromosomes found in the salivary glands of Drosophila , results from repeated rounds of DNA 2)replication without cell division known as Eudoreduplication.
3)Leads to about 500 copies of DNA for each chromosome
4)lie in alignment to produce very
if the polytene chromosomes are exposed to a thermal stress then
a discrete number of puffs are formed called chromosomal puffs, called Heat Shock. also relaxation of the chromatin structure as RNA transcription is initiated.
Chromatosomes are located at regular intervals along the DNA molecule and are separated from one another by
linker DNA
if a piece of chromatin contained 200 copies of the histone H4, then how many nucleosomes would be present?
100, it comes in pairs, plus histones are uniformerly spaced out.
nucleosomes consists of
8 histones proteins around which the DNA wraps 1.65 times
chromatosomes
consists of a nucleosome plus the H1 histone
how many histones associated with chromatosomes
9 b/c it includes the H1
how many copies of the H2B histone would be found in chromatin containing 50 nucleosomes?
100
Chromosomal Puffs
regions of relaxed chromatin structure as RNA transcription is initiated.
If a polytene chromosome is exposed to a thermal stress then
a discrete number of puffs are formed known as heat shock response
DNA associated with active gene expression
is more sensitive to degradation
DNA associated with protein is resistant to
degradation by DNAse
Chromatin shows tissue specific differences in
DNAse sensitivity
Polytene chromosomes are
rare, large unusual chromosomes that provide researchers with evidence of the changing nature of chromatin structure or organization by chromosomal puff visualizations.
Sensitivity of DNA to digestion of DNase I is correlated with
gene expression, suggesting that chromatin structure changes in the course of transcription of globin genes in erythroblasts of chick embryos.
centromere
is the attachment site for the spindle fibers during meiosis and mitosis
first centromeres studied at the molecular level were isolated from
Yeast
Yeast has unusually
small chromosomes, centromeres of most other eukaryotic cells are much larger.
typically 1,000s of copies of a short DNA sequence repeated in tandem, typically span more than 100,000 bp.
you have to use electron microscope to view this, which is the down
telomeres
ends of eukaryotic chromosomes, function as a cap to stabilize the end of the chromosome. the last few nucleotides of a linear DNA molecule cannot be synthesized. Would lead to shortening of the chromosome at each cell cycle.
replication of telomeres required a special enzyme called
telomerase
C's are always oriented towards the end of the chromosome. this is a?
telomeres
telomerase are structured with tandem repeat of a short DNA sequence
CCCTAA repeated 250-1500 times
all cancer cells have
an active telomerase bc telomerase is always active in (ACTIVELY DiVIDING Cells)
First artificial chromosome was built from
parts from yeast and a protozoan, but more recently is built from bacterial parts and mammals, each has a centromere, pair of telomeres and origin of replication.
large pieces of DNA can be
added and the artificial chromosome inserted into appropriate cell, used routinely for genetic engineering of large DNA fragments, several million bp in length which is important to the human genome project.
prokaryotic and eukaryotic cells differ greatly in the amount of
DNA per cell
c-value is
the amount of DNA per haploid cell
Drosophila has ___ times more DNA than ___.
35 times, E.Coli
Human cells have ___ times more DNA than ___?
10 times, Drosophila
but only twice as many genes
Some salamanders have ___ times more DNA than ___.
20 times, Humans
eukaryotic DNA has a complexity
that is not present in bacterial genomes
c-value paradox
c value refers to the amount of DNA in a haploid cell, when you compare how much DNA a human has compare to others like bacteria or Drosophila, this is a mystery, so that is why it's called a C-value Paradox.
when DsDNA is heated
H bonds break, and DNA separates into 2 ssDNA molecules called denaturation or melting.
Tm is
the thermal melting, temperature at which the double strand DNA is 50% single strand DNA.
Tm depends on the
DNA sequence
which base pairs are more stable GC or AT?
GC base pairs are more stable, bc melting GC base pairs requires more heat.
DNA with higher ( G+C ) content will have
higher Tm
denaturation is reversible by
slowly cooling the single stranded DNA, it will renature and reform the Double strand DNA.
Hybridization
anneal the complementary strands of DNA from different sources , usually 2 different organisms. Can form dsDNA if reasonably complementary, does not have to be perfect base for base match. and it produces recombinant DNA.
renaturation experiments showed that eukaryotic DNA
has 3 classes of DNA sequences, each having its unique sequence DNA which is only present only once or maybe a few times in the genome.
globin gene family
humans have seven different b globin genes grouped on chromosome 11, each associates with alpha globin polypeptides to make various forms of hemoglobin molecules. immunoglobulin gene family has several hundred members.
repetitive DNA
multiple copies of closely related sequences
moderately repetitive DNA
sequences from 150-300 bp repeated 1,000s of times, includes important genes like those coding for tRNA and rRNA. majority of moderately repetitive DNA sequences whose functions is just beginning to be understood
Tandemly repeated moderately repetitive DNA
repeat unit is clustered together and repeated one after another.
interspersed repetitive DNA
sequences are scattered throughout the genome
Alu sequences
200 bp and repeated at least 1 million times in the human genome, recent studies are beginning to give hints to function
SINEs are
short interspersed elements , alu repeats are an example.
LINEs are
long interspersed elements, mostly composed of transposons
Highly repetitive DNA
short 10 bp sequences repeated upwards of one million times or more, cluster around centromeres and telomeres, rarely transcribed and probably function to stabilize centromeres and telomeres.
gene density
density of functional genes varies widely among the chromosomes.
junk DNA
80% of all genomic DNA is regulated the importance gene expression
The genes for ribosomal RNA and transfer RNA make up which part of the 3 major classes of DNA
moderately repetitive DNA
transposable elements are
mobile elements, jumping genes, transposons and controlling elements , most commonly used name is transposons and transposable elements
transposons are abundant means
50% of the human genome is made up of transposons
transposons can be mutagenic means
insertional mutation by inserting into a key part of a gene, also promote DNA rearrangements ( inversions, duplications and deletions).
transposons are
abundant and ubiquitous
transposons have been found in
every organisms that has been investigated.
transposons general characteristics
varies greatly, but some common features include.
termini have short direct repeats, direct repeats are flanked by inverted repeats usually 9-40 bp long, enzyme needed for transposition recognizes the inverted repeat.
direct repeat results from the
mechanism for inserting into a chromosome. direct repeat will differ among various copies of the transposon, depending on the chromosomal site of insertion
transposon insertion
staggered cuts in target DNA by transposase. Direct repeat results from filing gaps in target DNA.
DNA transposons can be
replicative or nonreplicative
replicative
a new copy of the transposon is inserted into a new chromosomal site and the original copy is retained
nonreplicative
transposon excises from the old site and moves to a new site
> 50% of spontaneous mutations in drosophila are due to
insertion of a transposon in or near a functional gene
bar mutation is due to
duplication promoted by recombination b/w 2 transposable elements
drosophila transposons
a wide variety of transposons have been found in drosophila, p-elements has been studies extensively and used as a tool for molecular genetic studies.
P-elements
31 bp inverted repeats flanked by 8 bp direct repeats based on the genomic site of insertion
hybrid dysgenesis - males
males caught in the wild were brought back to the lab and mated with females of strains that had been maintained in the lab for a long time. F1 progeny were sterile or F2 showed new mutations and chromosome aberrations.
Hybrid dysgenesis - females
female flies from the wild were mated with males of strains that had been raised in the lab for many generations. The F1 progeny are fine as were the F2 generation.
Flies from the wild harbored
copies of the P element transposon
females with the P element transposon also produced
a repressor of transposition that were stored in the cytoplasm of the egg
lab strains of females lacked the
P-element transposon
without the repressor in the cytoplasm of the egg
the P element introduced by the males from the wild led to transposition and the resulting mutagenesis.
transposase was only active in the
germ line cells that is why you see that F1 progeny were sterile.
mutagenesis only occurred in
germ line of the F1 progeny, the f1 appeared normal but were either sterile or their progeny had serious mutations which is a result of differential splicing.
which is true of transposable elements?
1) they can promote DNA rearrangements
2) they often generate short flanking direct repeats
3) many posses short terminal inverted repeats
4) they are found in the genomes of all organisms.
In humans, transposable elements are
mostly inactive and no longer capable of transposition
The E.coli genome is
4.6 million bp (4.6 x 10^6 bp), in the lab e.coli can replicate in as little as 20 minutes
DNA replication must occur at a rate of
1,000 nucleotide per second, accuracy approaches one nucleotide error per 250 cells.
Conservative
2 new DNA duplexes would consist of 1 DNA duplex with both strands of "old" DNA from the original DNA and one DNA duplex of all "new" DNA
semiconservative
2 new DNA duplexes would each contain one DNA strand from the original DNA and one "new" strand of DNA
Dispersive
original DNA breaks down into fragments with "new" and "old" DNA interspersed with each other
Meselson and Stahl Experiment
designed an experiment to determine which of the 3 models was correct, needed to distinguish old from new. used 2 isotopes of nitrogen, could distinguish DNA containing one or both forms of nitrogen by differences in their densities.
equilibrium density gradient centrifugation
DNA samples are mixed with a heavy salt solution, usually cesium chloride, samples are placed in ultracentrifuge, centrifuged at very high speed for several days, gravitational forces > 100,000xg.
How does the centrifugation work
the cesium chloride forms a density gradient, DNA moves in the gradient until its density matches that of the gradient, heavier DNA moves closer to the bottom of the tube, DNA containing 15N.
If the dispersive model of DNA replication had been correct, Meselson and stahl would have observed that DNA extracted from bacterial cells following a second round of DNA replication in 14N would have been?
only of hybrid density, and somewhat lighter than after one round of replication.
In the density experiment you cannot distinguish
dispersive from semiconservative after the first round of replication. but after the second round of replication, dispersive would yield a single band with slightly lighter density.
theta replication
used mainly for replication of circular molecules, bacterial genomes and many plasmids
rolling circle replication
used by some viruses
Theta replication facts
named based on its resemblance to the greek letter theta, DNA strands begin to unwind at a DNA site called the replication origin, each single stranded DNA will serve as the template for synthesis of new DNA. Bidirectional replication is most common altho
the product of theta replication are
2 circular DNA molecules
DNA replication is
bi-directional
replication bubble
the unwinding of the double helix generates a loop, unwinding maybe at one or both ends of the bubble, making it progressively large.
replication fork
the point of unwinding where the 2 single nucleotide strands separate from the double stranded DNA helix
individual units of replication are called ___, each of which contains a ___.
replicons, replication origin
replication origin
starts at the origin, continues until the entire replicon has been replicated. bacterial have single replication origin, eukaryotes have many.
Rolling circle replication facts
produces multiple copies of the original DNA molecules, used by viruses bc each new viral particle needs its own copy of the viral genome. production line DNA replication.
conclusion of rolling circle replication
multiple circular DNA molecules, ssDNA circular is converted to dsDNA.
replication of eukaryotic DNA is more complex and proceeds
at a much slower rate b/c eukaryotic chromosomal DNA is larger than bacterial or viral genomes.
the product of eukaryotic DNA replication are
2 linear DNA molecules
DNA replication in eukaryotes differ from replication in prokaryotes in that?
eukaryotic chromosome have many separate origins of replication, where prokaryotic chromosomes have a single origin of replication.
DNA synthesis
nucleotides always added to the 3-oh on the newly synthesized DNA strand. the last 2 phosphate of the dNTP are cleaved, a phosphodiester bond is formed b/w 2 nucleotides.
replication requirements
ssDNA serve as template
substrates (dNTP) deoxyribonucleotides
primer
necessary enzymes (DNA polymerase)
replication direction
DNA polymerases are the enzymes that add nucleotides to only the 3'OH of the growing DNA chain, the 2 template DNA strands are antiparallel, DNA synthesis must proceed in opposite directions on the 2 template DNA strands.
Okazaki fragments
result of the short lengths of DNA produced by discontinuous replication of the lagging strand
lagging strand
the newly made strand that undergoes discontinuous replication
leading strand
proceeds continuously
discontinuous replication is a result of which property of DNA?
antiparallel nucleotide strands
OriC
is the DNA sequence where DNA synthesis is initiated in E. Coli.
Initiator proteins
bind to the oriC, DNA sequence and begin to unwind the DNA strands.
DNA helicases
continue the unwinding of the DNA that was started by the initiator proteins, and prevent the ssDNA from reforming dsDNA
single strand binding proteins
attach to the single stranded DNA, each SSB tetramer covers 35-65 bp, DNA sequence independent
DNA gyrase
a topoisomerse, reduces the torsional stress due to supercoiling ahead of the replication fork.
Primers
All known DNA polymerases require a primer to begin DNA synthesis, the primer provides the nucleotide with 3'OH that is needed to form the phosphodiester bond with the dNTP.
primase
synthesizes short RNA segments, on the DNA template, provides the 3'OH for DNA polymerase. these primers will have to be removed later.
leading strand facts
DNA synthesis is continuous, only requires 1 primer at the 5' end of the newly synthesized DNA
lagging strand facts
each okazaki fragment requires a primer, it has to have a primer, there will be thousands!
DNA polymerases in e.coli
consists of 5 of them, 1 and 3 are involved in DNA replication, other 3 involved in repairing damaged DNA.
DNA polymerase III in e. Coli
major polymerase used for DNA replication. large multi protein complex, major polymerase used for synthesis of genomic DNA.
5' to 3' polymerase
adds nucleotides to the newly synthesized DNA strand
3' to 5' exonuclease
can remove an incorrectly matched nucleotide, proofreading activity
DNA polymerase 3 is highly processive b/c
once DNA synthesis begins, DNA polymerase 3 can add many nucleotides without releasing from the template strand
DNA polymerase 1
first DNA polymerase to be isolated, unique in that it has a 5 to 3 exonuclease activity, used in one of the steps needed to convert the Okazaki DNA fragments on the lagging strand to a single DNA strand. 5'3 exonuclease is also used in repairing certain
DNA ligase
catalyzes the formation of a phosphodiester bond between the 5 phosphate to 3 OH of adjacent nucleotides
RNA primers must be ___ and the individual Okazaki fragments must be ___ together to form one DNA strand
removed, joined
primer functions
RNA primer removed by the 5 to 3 exonuclease activity of DNA polymerase I, the primer is replaced by DNA by the polymerase activity of DNA polymerase I, individual Okazaki fragments are joined together by DNA ligase.
Fidelity of DNA replication
overall accuracy of DNA synthesis is 1 error per billion nucleotides . its a proper pairing of the correct nucleotide by DNA polymerase.
proofreading
first level of checking, if the wrong nucleotide is inserted into the newly synthesized DNA strand, the polymerization process stops, two steps include, the 3 to 5 exonuclease removes the incorrect base, and the DNA polymerase activity inserts the correct
mismatch repair
last level of checking is that after DNA replication is complete, the mismatch DNA repair system checks for mismatched bases, mismatched bases are removed and replaced with the correct bases. process involves DNA methylation.
overall DNA replication in eukaryotic cells resembles that in bacterial cells, however there are additional complexities such as
greater size, multiple origins of replication, and DNA is linear instead of circular.
eukaryotic cells utilize 1,000s of replication origins, the entire genome must be copied without any sequences being duplicated or omitted. this process occurs in 2 steps.
first each origin is licensed for replication by the binding of a replication licensing factor, initiator proteins that begin the unwinding of the DNA at the replication origins only interact with the replication licensing factor. As DNA synthesis moves a
All DNA polymerases require a primer with a 3'OH group to begin DNA synthesis, the primer is:
a short stretch of RNA nucleotides
Eukaryotic DNA polymerases alpha
has the primes activity for synthesis of the RNA primer, continues with a short 30-40 DNA sequence
DNA polymerase Delta
finishes DNA synthesis on both the leading and lagging strands
DNA polymerase epsilon
involved in DNA replication in the nucleus on the leading strand
DNA polymerase gamma
used in replication of the mitochondrial DNA
DNA polymerase Beta
does not function in DNA replication, involved in DNA repair and genetic recombination.
Nucleosome Assembly
precise process of assembling newly synthesized DNA into chromatin is still not fully understood. Data suggest that newly assembled nucleosome are a random mixture of old and newly synthesized histones.
how does Eukaryotes fill in the gap?
telomerase extends the DNA, filling in the gap due to the removal of the RNA primer
telomerase facts
active in rapidly dividing cells
most mature somatic cells have little or no telomerase activity
tolomeres becomes progressively shorter
when the telomere is too short, the chromosome becomes unstable, leading to cell death
aging and cancer
genetically engineered mice that lack a functional telomerase gene have rapidly shorting telomeres, also shows many signs of premature aging. cells in culture that are engineered to always express the telomerase gene continue to divide in culture indefini
you have created a transgentic mouse that constitutively produces telomerase in all cells. you predict that these mice will
have longer telomeres than wild type mice of the same age. Have increased chance of developing cancer.
RNA facts
a polymer of ribonucleotides joined by phosphodiester bonds, contains ribonucleotides instead of deoxyribonucleotides, OH group on the 2' carbon, makes RNA less chemically stable than DNA
RNA contains
Uracil, whereas DNA contains thymine
RNA is ___ stranded
single
complementary regions within the single-stranded RNA can form
secondary structures such as hairpin-loops and stem-loops, in contrast DNA has fewer possible secondary structures and thus, a narrower range of cellular functions.
rRNA
important part of the ribosome, the site of polypeptide synthesis
mRNA
carries the genetic information encoding the sequence of amino acid for a polypeptide, provides the template for the ribosome to direct the order of amino acids in the polypeptide, eukaryotic mRNAs are synthesized as a larger pre-mRNA that requires extens
transfer RNA tRNA
brings the correct amino acid to the ribosome for addition to the nascent polypeptide chain
small nuclear RNAs snRNA
found in the nucleus associated with numerous proteins to form small nuclear ribonucleoproteins (snRNPs), pronounced snurps.
small nucleolar RNAs (snoRNAs)
involved in processing of rRNA in the nucleolus
small cytoplasmic RNA's (scRNAs)
found in the cytoplasm of eukaryotic cells, function is still not fully understood
MicroRNAs (miRNAs) and small interfering (siRNAs)
small RNAs in the cytoplasm of eukaryotic cells that interfere with translation of mRNA or initiate its degradation.
transcription
process of synthesizing RNA from a DNA template, overall transcription resembles DNA replication, but has key differences.
RNA molecules are
much shorter, transcription is carefully regulated to selectively transcribe specific gene at specific times in specific cell types, in higher eukaryotes, only a small portion of the genome is ever used for transcription
three major components of transcription
A DNA template, substrates, rNTPs usually called NTPs, proteins needed for transcription, RNA polymerase.
transcribed DNA strand
usually only 1 strand of dsDNA in a specific region will be used for transcription, DNA strand used for transcription is the template strand, other strand is the contemplate strand
no primer required for
RNA synthesis
RNA synthesis is always from the direction of
5' to 3'
non template strand
sense strand going 5' to 3
Gene a and C are, and B are
- strand, are transcribed from + strand
Transcription Unit
a length of DNA that contains all of the sequences needed for transcription
3 critical regions
promoter, RNA coding Region, Terminator
promoter
DNA sequence that the transcription apparatus recognizes and binds to in order to initiate transcription, Determines which DNA strand will be transcribed, also determines the start site, which corresponds to the first nucleotide in the RNA. usually adjace
RNA coding Region
actual DNA sequences that are copied into an RNA molecule.
Terminator
DNA sequence that signals the RNA polymerase to end transcription. terminator is usually part of the transcript since transcription ends after passing the terminator.
promoter is not
transcribed ever!
promoter is ___ of the transcriptional start site, transcription proceeds ___.
upstream, downstream
the nucleotide on the nontemplate DNA strand (sense strand) that corresponds to the first nucleotide of the RNA
designated +1
nucleotide downstream are assigned
positive numbers
nucleotides upstream are assigned
negative numbers
which of the following statement is not correct about process of transcription
during transcription the RNA molecule is synthesized in the 3' to 5' direction
substrate for transcription
RNA is synthesized from ribonucleotide triphosphates (rNtps or just NTPs), nucleotides are added to the 3-oh on the nascent RNA, 2 phosphates are cleaved from the nucleotide precursor, remaining phosphate is part of the phosphodiester bond, synthesis is t
Bacterial cells typically have
one RNA polymerase, responsible for synthesis of mRNA, tRNA, and rRNA, primers for DNA replication are synthesized by a different enzyme called the primes, large multimeric protein complex containing several polypeptides.
bacterial RNA polymerase core enzyme
2 copies of alpha subunit, 1 copy of beta and beta prime, and omega. not essential for activity but helps stabilize the enzyme complex.
core enzyme carries out the elongation
RNA by addition of nucleotides
sigma factor
directs the binding of the RNA polymerase to the promoter DNA sequence. called sigma for specificity.
addition of sigma to the core enzyme creates
holoenzyme, which is only required for promoter binding and initiation, sigma dissociates after addition of the first few nucleotides.
what would the likely result be if a specific sigma subunit were mutated?
RNA polymerase would fail to initiate transcription at the promoter specific to the sigma subunit.
3 distinct RNA polymerases
RNA polymerase 1 - transcribe rRNA
RNA polymerase 2 transcribes - pre-mRNA, snoRNA and some snRNA
RNA polymerase 3 - tRNA, small rRNA and some snRNA
each RNA polymerase is a large multimeric enzyme complex containing more than 12 polypeptide subunits.
3 stages of process bacterial transcription
initiation- transcription apparatus assembles on the promoter and RNA synthesis begins
Elongation-synthesis of the RNA as the transcription apparatus translocates along the mRNA
termination - RNA synthesis ends and the RNA separates from the DNA template
initiation
transcription apparatus must correctly interact with the promoter sequence of the DNA, primary step that regulates the rate of RNA synthesis, different genes are transcribed at different rates, mainly depends on the affinity of the transcription apparatus
bacterial promoter - the DNA sequence of the promoter determines
where transcription begin, which of the 2 DNA strands will be used as template, and direction that the RNA polymerase will move.
bacterial promoters are usually located adjacent
to the DNA that will be used as a template for RNA synthesis
bacterial promoters share a few short highly conserved DNA sequences, common nucleotide sequence is called a
consensus sequence
bacterial promoters are the most commonly
found nucleotide when a large number of promoter DNA sequences are examined.
consensus sequence conventions
BASES THAT occur with equal frequency are designated with a slash /.
Y - usually find a pyrimidine
R - usually find a purine
N - no base is more common than any other
exp: 5'-TAYARNA-3'
most commonly found sequence -35 consensus sequence
TATAAT
-35 nucleotides upstream of the transcription start site (+1)
TTGACA
most mutations in either the -10 or -35 sequence lead to slower initiation of transcription called
down mutations
some mutations make the rate of the transcription increase called
up mutations
in general, mutations that make the promoter more closely match the consensus sequence also cause
an increase in the rate of transcription
holoenzyme is the
core enzyme plus the sigma factor
sigma factor interacts with the
-10 and -35 sequence
RNA polymerase complex extends from
-50 to +20
binding of the holoenzyme to the promoter alters
its structure and allows it to begin unwinding the DNA
Upstream element
some bacterial promoters have a sequence of A-T pairs located from about -40 to -60, when present, the rate of transcription is increased significantly.
In a bacterial promoter, what binds to the -10 consensus sequence?
RNA polymerase holoenzyme (core enzyme + sigma)
RNA synthesis facts
a primer is not required, 2 of the 3 phosphates of the rNTP precursor are cleaved (as in DNA synthesis). 5' phosphate of the incoming nucleotide reacts with the 3'OH at the end of the nascent RNA to form a phosphodiester bond, sigma factor is released to
RNA polymerase unwinds
the DNA ahead of the complex and rewinds the DNA behind it. RNA synthesis is much slower than DNA synthesis.
unwinding the DNA creates positive
supercoils ahead of the transcription bubble and negative supercoils behind it. stress of supercoiling is relieved by topoisomerases.
unwinding of the helix
is the rate limiting step
transcription ends after
RNA Synthesis passes the terminator sequence, the terminator is transcribed into the RNA.
4 events must happen in termination
RNA synthesis stops, newly synthesized RNA is released from the RNA polymerase, RNA dissociated from the DNA template. RNA polymerase releases from the DNA template.
2 bacterial terminators
RHO-dependent termination, requires a protein factor called rho.
rho independent termination- termination does not required the rho protein, termination results from 2 common features encoded in the DNA
rho protein moves toward the
3' end of the transcript
rho protein has a
helices activity that unwinds the RNA from the DNA
Rho-independent terminations contains
an inverted repeat, transcription of the inverted repeat allows formation of a hairpin secondary structure. inverted repeat is followed by a series of 6 adenine residues. transcribed as a series of U's after hairpin structure.
transcription terminates when
inverted repeats form a hairpin followed by a string of uracils in a non-sense strand, and T's in a sense strand.
eukaryotic transcription differences
have 3 different RNA polymerase that each transcribe a different class of RNA and recognize different promoters. the interaction of the RNA polymerase with the promoter is more complex and involves numerous additional protein factors.
in eukaryotic cells, DNA is complexed with histone protein in a highly
condensed form, chromatin must be more realized to allow RNA polymerase to have access to the DNA, several proteins work together to alter the chromatin confirmation.
chromatin remodeling proteins
help remove nucleosomes away from promoters to facilitate interaction of the promoter with the transcriptional machinery.
histones
ARE BASIC POSITIVELY CHARGED AMINO ACIDS
2 classes of initiation regulatory sequences
promoters, enhancers
promoters in eukaryotes
always located adjured to the gene it regulates, promoters for RNA polymerase 3 are located within the gene. location is fixed relative to the transcription start site.
enhancers in eukaryotes
not always located near the transcription start site (+1), can function when located 1,000's of bases from the transcription start site. position can vary, can be located upstream or downstream of the gene or even within an intron.
no sigma factors in eukaryotes
instead its accessory proteins called transcription factors that recognize the promoter and then attract the appropriate RNA polymerase.
general transcription factors
interact with RNA polymerase to form the basal transcription apparatus. assembly is near the transcription start site and a low level of transcription can begin.
transcriptional activator proteins
bind to specific DNA sequences and stimulate higher levels of transcription by stimulating a faster rate of assembly of the basal transcription apparatus.
RNA polymerase 2
RNA polymerase that transcribes genes that encode proteins, the promoter recognized by RNA polymerase 2 has parts, core promoter and regulatory promoter.
Core promoter
located immediately upstream of the transcription start site , typically have consensus sequence called the TATA box, TATAAA located from -25 to -35, mutations alter the efficiency of transcription. changing its location alters where transcription begins.
regulatory promoter
located upstream of the core promoter, a variety of different consensus sequences have been found, the regulatory promoters are used in different combinations for different genes. transcriptional activator proteins bind to these sequences, which interact
RNA polymerase 1 and 3 promoters process is very similar to
polymerase 2 promoters, except each RNA polymerase recognizes different promoters and uses different general transcription factors.
promoters of RNA polymerase 2 are unusual because they are
located downstream of the transcription start site and are actually transcribed as part of the RNA.
TFII
transcription factor for RNA polymerase II
TFIID
binds to the TATA box of the promoter, which places the active site of RNA ploymerase II in the correct place to begin transcription of the piper nucleotide (+1). TFIID is a complex of at least 9 polypeptides. One polypeptides is the TATA box binding prot
TATA box binding protein (TBP)
binds to the minor grove of the DNA and bends the DNA which begins to unwind the 2 DNA strands
This fly is homozygous for a mutation in the fru gene. what is the sexual orientation of this fly?
bisexual
gene
a set of nucleotides in DNA that specifies the order of amino acids of a protein
genes and proteins are said to be
colinear
francis crick was the first to propose that there is a
direct relationship b/w the DNA sequence and amino acid sequence of a protein
with colinearity, the number of
nucleotides in the gene is proportional to the number of amino acids in the protein, for bacterial and viral genes, this assumption is fairly accurate. but eukaryotic genes have a very different structure
R looping
use electron microscopy and visual RNA and DNA molecules, this technique can look a thickness, you can distinguish single from double stranded. The conclusion is the coding sequences can be interrupted by non coding sequences. Non coding regions of DNA ap
exons
coding regions, non coding regions are introns
genomic DNA with the R looping represents one of the
introns
eukaryotic genes typically have regions of DNA that are transcribed and appear in the mature RNA called
exons (expressed sequence)
eukaryotic genes also have regions of DNA whose sequences are not part of the mature RNA called
introns (intervening sequences)
intron number and size increase with increasing organismal complexity
for example, yeast has less introns than drosophila
group 1 introns
found in some RNA, self splicing, catalytic RNA
group 2 introns
found in some protein coding genes in the mitochondria, chloroplast and a few bacteria, self-splicing but use a different mechanism than group 1 introns
nuclear pre-mRNA introns
found in the protein coding genes in the nuclear genome, mechanism is similar to that of group 2 introns, but it requires a complex of snRNAs and proteins called a spliceosome
transfer RNA introns
utilize yet another splicing mechanism that relies on proteins
transcription unit facts
contains the promoter, RNA coding sequences and terminator, the RNA coding sequence includes all the exons and introns. remember that a gene product can be RNA, tRNA, rRNA, snRNA
Messenger RNA
DNA cannot directly encode the information for synthesis of protein since the DNA is in the nucleus and protein synthesis occurs in the cytoplasm
the newly discovered RNA was called messenger RNA since it
carried the genetic info for the amino acid sequence of the protein from the nucleus to the ribosomes in the cytoplasm.
there are 3 primary regions to the mRNA of both prokaryotes and eukaryotes
5' untranslated region (5'UTR) -leader sequence
protein coding region - open reading frame (ORF)
3' untranslated Region (3'UTR) - does not code for amino acids
5' untranslated region (5'UTR)
leader sequence, does not code for amino acids, in bacteria the 5'UTR contains Shine Dalgarno Sequence. Ribosome biding site.
protein coding region
usually called the open reading frame (ORF), beings with a start codon, all prokaryotic and eukaryotic proteins begin with AUG, which codes for methionine. ends with one of 3 stop codons.
3' untranslated region (3'UTR)
does not code for amino acids, primarily affects the stability of the MRNA.
Jr. Scientist has identified sequences on an mRNA from a vampire bat that she thinks are important for proper association of the mRNA with a ribosome. these sequences must be part of which portion of mRNA.
5'Untranslated Region (bc of the association of ribozymes)
in prokaryotic cells, transcription (RNA synthesis) and translation (protein synthesis) occurs
simultaneously.
ribosomes can attach to the shine-dalgarno sequence near the 5'end even before transcription reaches the terminator sequence. bacterial mRNA is not modified before being used for translation
transcription occurs in the ____, translation occurs in ____.
nucleus, cytoplasm (in eukaryotes only)
eukaryotic mRNA requires significant
modifications after synthesis of the pre-mRNA before it is suitable for translation, changes occur at the 5'end, 3'end and the protein coding sections.
5'cap
a modified guanine nucleotide is added to the 5'end of the RNA. going 5' to 5'.
it has all 3 phosphates
methyl at position 7 base
methyl groups is added to the 2'oh of one or few nucleotides at the 5' end. cap is required for translation, cap also affects
polyadenylation
a series of 50-250 "A" nucleotides are added to the 3' end of the RNA. there is not a corresponding series of "T's" in the template DNA.
adenine nucleotides are added by an enzyme complex containing a number proteins including
polyadenylate polymerase
poly (A) tails affect
stability of the mRNA and binding of the mRNA to the ribosomes
histone mRNA are unique in that they lack
the poly (A) tail
RNA splicing
removal of the introns, usually occurs after capping and polyadenylation, but before the RNA is transported to the cytoplasm.
3 sequences are needed for splicing
5' splice site - GU consensus sequence
3' splice site -AG consensus sequence
branch point - located 18-40 nucleotides upstream of the 3' splice site
YNYYRAY - consensus sequence
mutation in the GU (5'splice site), AG (3'splice site) or A (in the brand point) all prevent
splicing
splicesome
large complex containing several protein and RNA's, small nuclear RNA (snRNA) in the splice some are 107-210 nt in length. associate with the proteins to form small nucleoprotein particles called snurps.
1st step of splicing
1st - premRNA is cleaved at the 5'splice site , exon 1 is free of intron, 5'end of the intron attaches to the A in the branch point, forms a structure that looks like a lariat. novel 5' to 2' phosphodiester bond b/w the G at the 5'end of the intron and A
second step in splicing
RNA is cleaved at the 3' splice site, simultaneously the 3'end of exon 1 is covalently joined to the 5'end of exon 2. intron is released as a lariat. the bond at the branch point breaks. the intron becomes linear and is degraded by nuclear nucleases.
splicing of nuclear pre-mRNA requires
splicesome, involves interaction b/e mRNA and the SnRNAs. key point - the snRNAs are complementary to different parts of the mRNA. this allows critical parts of the pre-mRNA and the splicesome to come close enough for the splicing reactions to occur.
nuclear organization
conventional thought held that nucleus and cytoplasm lacked structure and was a biochemical soup. recent studies show that nucleus and cytoplasm have an ordered structure. RNA synthesis and splicing occurs within discreet regions of the nucleus.
group 1 and 2 introns are
self splicing, no proteins needed. RNA has a true enzymatic activity to catalyze the splicing reaction
group 1 introns are commonly found in
some rRNA gene of protozoans, mitochondrial genes of some fungi and very recently found in few bacteriophage genes.
group 2 introns are usually found in
mitochondiral genes
the enzymatic activity of self splicing RNA depends on the complex secondary structure of
RNA, catalytic RNA is commonly called a ribozyme.
Single pre-mRNA can be processed to yield different mRNA molecules, different polypeptides can be derived from a single gene commonly called
isoforms
Multiple 3' Cleavage sites
another form of alternative splicing, utilizes multiple sites for 3' cleavage of the pre-mRNA and polyadenylation. May not always produce different polypeptides since the cleave sites may be downstream of the stop codon of the open reading frame (ORF).
Importance of alternative splicing
estimated that up to 60% of human genes have isoforms due to alternative splicing. Many genetic disease are mutations that affect splicing. 15% of single-base mutations leading to a human genetic disease result in splicing errors. some mutations actually
the coding sequence of the mRNA is changed after transcription
changes includes insertion and deletion of bases as well as nucleotide changes, and there are 2 major mechanisms.
guide RNA (gRNA) is partially complementary to the RNA to be edited
guide RNA adds nucleotides to the pre-mRNA that were not encoded by the DNA.
second mechanism involves enzymes that change the nucleotides in the mRNA.
for example, cytosine can be converted to uracil by removal of an amine (NH3) group by deamination. Base change can alter a codon to a stop codon. result is a shortened polypeptide.
transfer RNA (tRNA)
tRNA is the adapter that joins the amino acid to the corresponding codon of the mRNA. each tRNA is specific for one of the 20 amino acids found in proteins. most organisms have 30-40 or more tRNAs, each encoded by a different gene.
tRNA contains
4 usual nucleotides (A, U, G, and C), also contains numerous modified bases that result from enzymatic reactions after the tRNA is synthesized.
Most tRNA are transcribed by
polymerase 3 (eukaryotic cells) as a larger precursor containing several tRNA genes. cleaved, chemically modified and trimmed to produce the mature tRNA. some tRNA precursors have introns, splicing mechanism is quite different than that of spliceosomes.
in bacteria , rRNA genes are
dispersed throughout the genome
in eukaryotes, the multiple copies of the rRNA genes are
clustered together
three of the 4 rRNA genes are transcribed as a single precursor by
RNA polymerase I, processed to yield the mature rRNAs.
the 5s rRNA is transcribed by RNA
polymerase 3 and is encoded by a separate gene
small RNAs
double stranded RNA complementary to a specific mRNA would inhibit translation when injected into cells. lead to discovery of 3 classes of small RNA molecules.
small interfering RNA (si RNA)
microRNA
Piwi-interacting
RNA interference
evolved as a protective mechanism against viruses and transposons. RNA interference is triggered by double stranded RNA molecules (dsRNA). Transcription of inverted repeats so the RNA base pairs with itself. transcription of 2 different RNA molecules that
dsRNA is chopped up by an enzyme called
Dicer
unwinding of the small dsRNA produce
siRNA and miRNA
RNA combines with proteins to form an
RNA-induced silencing complex (RISC)
proteins have numerous important roles in the cell
enzymes, structural components, regulation, defense, communication
H3N and COO stands for
Amino and carboxyl group
H and R stands for
Hydrogen and R radical group side chain
GLY, G
Glycine
Ala, A
Alanine
Val, V
Valine
Leu, L
Leucine
IIe, I
Isoleucine
Met, M
Methionine
Phe, F
Phenylalanine
Try, Y
tryosine
Trp, W
tryptophan
Ser, S
Serine
Thr, T
Threonine
Cys, C
Cysteine
Pro, P
Proline
Asn, N
Asparagine
Gln, Q
glutamine
Lys, K
Lysine (positively charged R groups)
Arg, R
Arginine (positively charged R groups)
His, H
Histidine (positively charged R groups)
Asp, D
Aspartate (negatively charged R groups)
Glu, E
Glutamate (negatively charged R groups)
carboxyl group of one amino acids forms a ____ with the amino group of the next amino acid
peptide bond
______ are a linear polymer of amino acids .
polypeptides
polypeptides have polarity, one end has a free ____ group and the other end has a free ___.
amino, carboxyl
polypeptides have several levels of organization known as
primary, secondary, tertiary, quaternary
primary structure
sequence of amino acids
secondary structure
beta pleated sheets and alpha helix
tertiary structure
overall three dimensional shape
quaternary structure
2 or more polypeptides interact to yield the final protein
a group of 3 nucleotides that specify either an amino acid or a translation stop signal is a
Codon
there are ___ possible condons
64
how many codons code for amino acids
61
an amino acid can be coded for by 2 or more codons known as ____, and 2 examples are?
degenerate code
tryptophan (W) and methionine (m) each have one codon
some amino acids have as many as 6 codons called
synonymous
Isoaccepting tRNAs
different tRNAs that use the same amino acids but have different anticodons, most organisms only use 30-50 of the possible 61 tRNAs.
Wobble
usually, synonymous codons differ in the 3rd position, first and second base of the codon strictly follow standard base pairing rules. third base of the codon is a little sloppy with its pairing. some tRNA can pair with more than 1 codon.
Any RNA sequence has 3 potential ____
reading frames
the same RNA sequence specifies 3 very different amino acids by changing which
nucleotide begins the reading frame
Initiation codon establishes the reading frame also called
start codon, always AUG
recently found a few examples where ___ and ___ are used as start codon
GUG, UUG
In bacteria, a modified methionine called ___ is used to begin each polypeptide
N-formylmethionine
three codons signal the end of polypeptide synthesis UAA, UAG, UGA
called stop codons, termination codons or nonsense codons ( no tRNAs with these codons), release factors interact with these codons.
genetic code is almost universal among prokaryotes and eukaryotes with a few exceptions
usually involve the stop codons, a few affect a sense codon, most of the exceptions are in genes in the mitochondrial genome.
protein synthesis occurs on the
ribosomes
the ribosomes attaches near the ___ of the mRNA and then moves towards the ___.
5'end, 3'end
Translation involves 4 steps
tRNA charging, Initiation, Elongation, Termination
tRNA charging
amino acid is attached to the correct tRNA
Initiation -
assembly of the mRNA and all necessary factors to the ribosome
Elongation
synthesis of the polypeptide by sequential addition of amino acids
Termination -
synthesis stops, the polypeptide and ribosome are released.
the enzymes that add the amino acid to the proper tRNA are called
aminoacyl tRNA synthetases
eukaryptic mRNA does not have an ____ that facilitates attachment of the small subunit
analogous Shine-Dalgarno
A consensus sequence surrounds the start codon to make the process more efficient called
Kozak sequence
In elongation, ribosome has 3 sites for tRNA
aminoacyl (A) site
Peptidyl (P) site
Exit (E)
also requires elongation factor Tu (EF-Tu), elongation factor Ts (EF-Ts) and GTP in bacterial cells.
peptide bond forms between the amino acid in the
A and P sites
TRNA that was in the ___ site is released via the ___ site. tRNA in the ___ site moves to the ____ site.
P site, E site, A site , P site
at the end of each cycle of elongation, the amino acids that was in the ___ site, is added to the polypeptide chain and ____ site is free to accept another tRNA.
A, A
Peptide bond formation requires
peptidyl transferase
release factors recognize the
3 stop codons, in e.coli there is one release factor for each stop codon
key differences b/w eukaryotic and prokaryotic cells
in bacteria, f-MET is used for initiation, transcription and translation occur simultaneously in prokaryotic cells. mRNA tends to be short lived in bacterial cells, usually only a few minutes. Eukaryotic mRNA can often remain for hours to days. Ribosomal
in both prokaryotes and eukaryotes, the newly synthesized polypeptide requires additional modifications before it is function like
chemical modifications such as Acetylation, Mehtylation, phosphorylation, glycosylation (addition of carbohydrate groups). some amino acids may be removed.
proper protein function requires folding into the precise 3 dimensional structure, some proteins fold properly on their own, some requires special proteins called
molecular chaperones
many antibiotics have been designed to interfere with various aspects of ___ in bacterial cells.
translation, and this is effective b/c ribosomal proteins rRNA, factors in bacterial cells are quite different from those of eukaryotic cells
tetracycline
blocks entry of tRNA into the A site of the ribosome
Chloramphenicol
binds to the large subunit and blocks peptide bond formation
streptomycin
binds to the small subunit and blocks initiation
erythromycin
blocks translocation of the ribosome to the next codon
A single gene can yield multiple types of mRNA by all of the following mechanisms except
shuffling the order of the exons in the mRNA relative to their order in the DNA
if instead of 20 amino acids there were 200 amino acids, what would you predict would be the minimum number of bases in a codon
4
what is the first nucleotide at the 5' end of the anticodon
G
suppose u have identified segment of genomic DNA that contains the YFG locus. How many possible reading frames are there for the ORF of the mRNA?
6 (until you know which is sense and the nonsense, you need to think of it as 6 reading frames)
sense codons code for ____?
amino acids
theres tRNA only for the
61 codons not 64
Bacterial cells responds to their environment
rapid changes in the environment lead to rapid biochemical changes within the bacterial cell, occurs through regulation of gene expression
Gene is any
DNA sequence that is transcribed into an RNA
structural Genes
encode proteins needed for cellular processes such as metabolism, biosynthesis, structure, hormone, etc.
regulatory genes
have products that are either RNA or protein, which function in controlling transcription or translation. Many are DNA binding proteins. affect the expression of the genes to which they are physically linked. many proteins products of regulatory genes bin
Regulatory elements are
DNA sequences that are not transcribed into RNA.
major step in regulating gene expression in both prokaryotic and eukaryotic cells is the
initiation of transcription
regulatory proteins often have characteristic
protein domains
protein domains are
usually 60-90 amino acids, of which only a few actually contact the DNA
simple protein structures that are frequently found associated with DNA binding proteins, common ones include
helix loop helix
zinc fingers
leucine zipper
Helix-turn -Helix
2 alpha helices connected by a turn
Zinc fingers
loop of amino acids associated with a zinc ion. often find several zinc fingers in a DNA-binding proteins. Fingers fit into the major groove of the DNA helix
leucine zipper
helix of leucine residues and an arm of basic amino acids
major differences in gene organization in bacterial and eukaryotic cells
in bacteria, genes with related functions are clustered together and often transcribed into a single mRNA-Operon
In eukaryotes, genes with related functions are typically scattered throughout the genome and each is expressed as a separate mRNA.
regulatory gene is not part of the operon
its protein product is needed for regulating expressed of genes in the operon, transcribed and translated into a protein called the regulatory protein
Operator is a DNA sequence that is part of the operon
the regulatory protein binds to the operator DNA sequence and affects transcription of the structural genes
Does this picture depict polyribosomes in prokaryotic or eukaryotic?
prokaryotic b/c of transcription and translation.
4 types of gene expression
negative inducible
negative repressible
positive inducible
positive repressible
negative inducible operons
the regulatory protein is a repressor
binding of the repressor protein to the operator blocks transcription
inducible because something must happen to prevent binding of the repressor protein
transcription of the structural genes proceeds when the repress
when the inducers is present
it binds to the regulator, thereby making the regulator unable to bind to the operator. transcription takes places. Binding of the inducer to the repressor protein causes an allosteric change (shape change) and the repressor-inducer complex cannot bind to
negative repressible operon
repressible means the gene is transcribed unless something acts to repress (inhibit) it. regulator protein is inactive until the corepressor binds to it. Allosteric change allows the now active repressor to bind to the operator.
which of the following is not a correct description of how translation differs bw bacteria and eukaryotic cells?
esr
eukaryotic and prokaryotic have how many release factors each?
1 for eukaryotic
2 for prokaryotic
A ____ operon is normally turned on, and it is turned off by a _____?
repressible; repressor
lactose must be transported across the cell membrane requires a
permease (lac Y)
lactose must be broken into glucose and galactose
requires b-galactosidase lacZ
a thiogalctoside transacetylase lacA gene is also made
but the function is still unknown, these 3 genes are structural genes
all these genes transcribed as a single mRNA encoding the 3 proteins called
polycistronic
in the absence of lactose, the regulator protein a repressor
binds to the operator and inhibits transcription
lactose is present, it is converted to allolactose by b-galactosidase (Bgal).
allolactose is the inducer for the lac repressor
the lac operon is repressed in the absence of lactose, permease is needed to transport lactose into the cell, how does the lactose get into the cells if there is no permease?
repression is never 100%, cells always have very low levels of the 3 proteins perm ease, b galactosidase, and the transacetylase, when lactose is added to the medium, the perm ease transports a small amount of lactose into the cell and b gal converts a sm
Low levels of glucose lead to all of the following except
cap-cAMP loses its affinity to DNA
the lac operon is an example of a negative inducible genetic system b/c
the binding of the lac repressor to the operator inhibits transcription by RNA polymerase
positive control and catabolite repression
when given a choice, E. coli and other bacteria prefer glucose over other sugars, such as lactose, glucose can enter glycolysis pathway directly. if glucose is present, metabolism of other sugars in the medium is repressed. called catabolite repression.
a form of positive control by this protein
catabolite activator protein, binds to a 22 nucleotide DNA sequence located slightly upstream and overlaps the promoter for the lac genes. key point - RNA polymerase binds much more efficiently if CAP is already bound to the promoter.
CAP must form a complex with
adenosine 3', 5' cyclic
Monophosphate (cyclic AMP) before it can bind to the lac promoter, often called a second messenger.
Cyclic AMP levels are inversely proportional to glucose concentration. High concentrations of glucose lowers the concentration of c
polycistronic mRNA
one mrNA codes for all 3 polypeptides
low levels of glucose lead to all of the following
cAMP levels increase, perm ease levels increase, B galactosidase level increase, the lac operon is activated, which mean it is ready for transcription of lactose to lactase.
trp operon
contains the genes that code for the enzymes for synthesis of tryptophan, five polypeptides that form 3 enzymes, two enzymes contains 2 polypeptides each. repressible system - transcription is normally on unless something acts to slow it down.
attenuation
form of gene regulation that couples transcription and translation. functions to slow expression of the genes need for tryptophan synthesis as the levels of tryptophan in the cell increases, called feedback inhibition. not found in eukaryotic cells since
Bacterial chromosome consists of?
1) larger circular DNA that is a series of twisted loops
2) appear as a distinct clump called the nucleoid
3) bacterial DNA not attached to Histones like Eukaryotes
relaxed state of DNA is
100 bp of DNA, about 10 complete turns, anything over is over rotated, and anything below is under rotated. So a DNA molecule with 300 bp long has 20 complete rotations when its suppose to have 30, its consider negatively supercoiled which is under-rotate
Since Eukaryotic DNA is associated with proteins, this is known as Chromatin. The two basic types of chromatin are?
Euchromatin - normal process of condensation and decondensation in the cell cyle
Heterochromatin - remains in a highly condensed state throughout the cell cycle event during interphase.
The major difference b/w chromatin and heterochromatin is that
transcription takes place in chromosomal material consisting of euchromatin. while heterochromatin is just for the centromeres and telomeres.
what's abundant in a chromatin?
proteins called histones - 5 types
it's positively charged, so that it can attached itself to the negative phosphate on DNA
neutralizing the histone charges from positive to neutral would cause?
histones to be separated from DNA
the levels of chromosome from large to small
chromosome
chromatin
Chromatosomes - nucleosome + H1
Nucleosomes 8 histones
30 nm fiber is the
solenoid
Polytene chromosome is what and how it happens
its a giant chromosome found in Drosphila, very unusual because its caused by repeated rounds of DNA replication, but no cell divisions.
D Nase I ?
an enzyme that digest DNA, and when DNA is unbound by histones, its more easily digested and sensitive to this enzyme. In conclusion, when genes become transcriptionally active, they also become sensitive to DNAse I, indicating the chromatin structure is
What is the nature of the change in chromatin structure that produces chromosome puffs and DNAse I sensitivity?
acetylation, methylation, phosphorylation all loosen the histone grip on DNA.
Epigenetic changes are alterations of
chromatin or DNA structure that do not include changes in the base sequence but are stable and passed on to cells or organisms. some epigenetic changes result from alterations of histone proteins.
most centromeres are made up of?
heterochromatin
What happens to a chromosome that loses its centromere?
will not segregate into the nucleus in mitosis and is usually lost.
What is a characteristic of DNA sequence at the telomeres?
they consist of guanine and adenine ( or thymine) nucleotides.
they consists of repeated sequences
one strand protrudes beyond the other, creating some single stranded DNA at the end.
2 single stranded molecules of DNA from different sources, such as different organisms will anneal if they are complementary, a process termed
hybridization
Eukaryotic DNA comprise three major classes ; unique-sequence DNA, moderately repetitive DNA, and highly repetitive DNA. Most of the Genes that encode proteins are found in
unique - sequence DNA
____ are generated when a transposable element inserts into DNA.
flanking direct repeats
transposable elements are mobile DNA sequences that often cause
mutations. there are many different types of transposable elements; most generate short flanking direct repeats at the target sites as they insert. many transposable elements also posses short terminal inverted repeats.
how are flanking direct repeats created in transposition?
in transposition, staggered cuts are made in DNA and the transposable element inserts into the cut. later replication of the single stranded pieces of DNA creates short flanking direct repeats on either side of the inserted transposable element.
transposition through an RNA intermediate
requires reverse transcription to integrate into the target site.
Which type of transposable element possesses terminal inverted repeats
all of them
insertion sequence, composite transposons, noncomposite transposons
hybrid dysgenesis results from?
a male fly with P elements (P+) mates with a female fly that lacks p elements (P-)
Which type of replication requires a break in the nucleotide strand to get started?
rolling - circle replication, new nucleotides are added to the 3'end of the broken strand, with the inner unbroken strand used as a template.
DNA polymerase 3
synthesize new DNA on the leading and lagging strand
DNA polymerase 1
removes and replaces primers
DNA ligase does what
seals the nicks that remain in the sugar-phosphate backbones when the RNA primers are replaced by DNA nucleotides.
The ends of the eukaryotic chromosomes are replicated by
an RNA protein enzyme called telomerase. this enzyme adds extra nucleotides to the G-Rich DNA strand of the telomere.
what would be the result if an organism's telomerase were mutated and nonfunctional?
chromosomes would shorten with each new generation.