Promoter
regulatory DNA sequence, usually at the upstream (5') region of the gene
Coding sequence
stipulates the amino acid sequence
Consensus sequences
conserved in nature
- critical role in biological processes
Termination sequence
marks the end of a gene
The synthesis of proteins requires what?
transcription and translation
What type of genes only get transcribed?
various types of RNA
-rRNA, tRNA, snRNA, etc
What is the Central Dogma of Genetics?
information contained in genes is decoded to produce other molecules that determine the phenotypic traits of organisms
What are the processes of the Central Dogma of Genetics?
DNA Replication:DNA to DNA
Transcription: DNA to mRNA
Translation: mRNA to protein
promoter region
A regulatory region a short distance upstream from the 5' end of a transcription start site that acts as the binding site for RNA polymerase. A region of DNA to which RNA polymerase binds in order to initiate transcription.
start codon
AUG (ATG in DNA)
What do promoters do?
show DNA polymerase exactly where to begin making RNA (they are signals), there are also signals to show when to stop
Important prokaryotic promoters
TATAAT -(10 region) 'Pribnow box'
TTGACA - (-35 region)
Important eukaryotic promoters
TATAAAA - (-30 region) - 'TATA box'
GGCCAATCT - (-80 region) - 'CAAT box'
typical promoter is 2kb long
Purpose of the coding region
to spell out the sequences of amino acids
What is the coding region based on?
a codon triplet which is
3 nucleotides = amino acid
4^3 (=64) triplets, 20 amino acids
Degenerate code
More codons than are necessary, exemplified by the amount of triplets that can be created (4^3=64)
What other sequence is the coding sequence a part of?
the transcribed sequence
What are the typical stop codons?
UAA, UAG, UGA
What does the transcribed region contain?
The coding sequence, but it is broader than just this and includes:
-transcription start site, coding sequence, start of the termination site
-full termination site expands further
all known proteins start with what letter?
M.
out of the 64 codons ___ code for amino acids while the other three are...
61 code for amino acid (one of these, AUG is an amino acid and a start codon) , 3 encode stop codons
Exceptions to the genetic code
There are changes in meaning for certain codons which depend on different types of species, many are changes with stop codons
What is the wobble hypothesis?
When multiple codons specify the same amino acids, most of the codons will have the same first two letters and there is flexible base pairing at the third position
Proline and the wobble hypothesis example
CCU
CCC
CCA
CCG
all have a different 3rd position but still code for proline
exons vs. introns
exons are coding segments
introns are non-coding segments that are transcribed but then spliced out
(INtrons stay IN the nucleus, whereas EXons EXit and are EXpressed)
do prokaryotic mRNA molecules have introns?
not typically, they can be polycistronic which means multiple genes are represented in one mRNA and transcribed from one promoter, they are then quickly translated because there is no need for transport out of a nucleus (lacking)
Does transcription end immediately after the STOP codon?
no, ends beyond it, genetic code ends here
What is a UTR?
untranslated region of the mature mRNA that will not be translated into proteins but have other functions such as attachment during translation.
Eukaryotic vs prokaryotic genomes
Eukaryotic genomes are larger, encode SINGLE proteins or mRNA while prokaryotes can have multiple genes on one promoter
Prokaryotic genes
Genes are in tandem.
Functions are related.
Genes are expressed together.
Operon
group of genes operating together and linked in a single unit
Viral gene structure
efficient, some viral DNA sera can encode multiple genes
DNA serves as a template for____
RNA transcription
which 4 ribonucleotides is RNA synthesized from
A, U, G and C
why is primer not required for RNA polymerase?
RNA polymerase enzymes are large
RNA
Make up ribosomes (along with ribosomal protein subunits).
Site of protein assembly
mRNA
-CARRY (messenger) coding instructions from DNA to ribosomes.
Specifies the sequence of amino acids.
mRNA is derived from pre-mRNA after extensive processing
tRNA
-Translation.
Brings in an amino acid to match the coding triplet in the mRNA to elongate the polypeptide chain
snRNA
Combine with small nuclear protein subunits to form small nuclear ribonucleoproteins (snRNPs - known as "snurps")
Process pre-mRNA into mRNA
miRNA and siRNA
act on mature mRNAs in the cytoplasm to block translation and mediate turnover of selected mRNAs
Ribozymes
RNA molecules that function as enzymes
which region of DNA coding for RNA determines the transcription start site?
the promoter which also determines which strands to transcribe
RNA-coding region
DNA sequence copied into RNA
Terminator
DNA sequence that signals for transcription to end, occurs after the terminator sequence has been copied.
On how many DNA strands does transcription take place?
only one
On how many DNA strands does replication take place?
both
Template strand
DNA strand used for transcription, but all genes do not necessarily reside on one strand
Nontemplate Strand
not transcribed
RNA molecule formed in transcription is____ to the template DNA strand
complementary, has the same sequence as the non-template strand except U instead of T
In which direction does transcription proceed?
5' to 3'
in prokaryotes, is a primer required for transcription?
no
without the sigma factor, what happens?
RNA polymerase cannot transcribe
sigma factor
controls the binding of RNA polymerase to the promoter
rho factor
facilitate transcription termination
How many subunits of RNA poly are there and what are they?
5 subunits that catalyze elongation
Alpha-? (2), beta-?, beta prime-?', and omega-?
transcription in bacterial RNA
all transcription steps are carried out by RNA polymerase unlike in replication
Holoenzyme
enzyme and a coenzyme
Prokaryotic promoters
TATAAT - located 10 bases upstream of the transcription start site (-10 region, 'Pribnow box').
TTGACA - (-35 region)
elongation in prokaryotes
? subunit dissociates, RNA poly moves downstream, unwinding, supercoiling occurs
Topoisomerase enzymes
relieve tension due to supercoiling
Termination in prokaryotes
occurs when a sequence is encountered (like a stop codon) that indicates termination, either rho dependent or independent
Rho dependent termination
Able to cause termination only in the presence of rho factor protein
Rho independent termination
intrinsic termination, able to terminate without rho factor
two components of rho independent termination
1. Inverted repeats that, when transcribed into RNA, form a hairpin loop
2. Six adenine nucleotides
Main differences in transcription between pro/euks
Multiple RNA polymerases in euks
Nucleosome structure
Promoter binding and initiation differs
How many types of RNA poly are there and what are they?
RNA poly 1,2 and 3
1. rRNA
2. pre-mRNAs, snoRNAs, and snRNAs (messenger rnas)
3. tRNA
Is chromatin remodeling necessary in pro or euks?
eukaryotes, this is needed to access the DNA
Acetyltransferase protein
adds acetyl groups to amino acid ends of histone proteins, destabilizes the nucleosome structure
Accessory proteins in eukaryotic transcription
General transcription factors and Transcriptional activator proteins
General transcription factors
Form basal transcription apparatus with RNA polymerase
What does Basal refer to?
enabling a low level of transcription
Transcriptional activator proteins
bind to specific recognition sequences
RNA poly 2 promoters
the promoter, the core promoter and the regulatory promoter
Core promoter
Immediately upstream of coding sequence
TATA box: -30 bp
TFIIB recognition element: -35 bp
Regulatory promoter
Immediately upstream of core promoter
Specific recognition sequences: CAAT box, GC box, OCT box
Enhancers
Further away from transcription start site (>2kb), sequences that increase rate of transcription, required for full level transcription
Initiation in eukaryotes
begins at consensus sequence upstream of the AUG start codon, Kozak in euks
Elongation in eukaryotes
same as in bacteria, RNA Polymerase leaves the promoter & transcription factors then moves downstream. RNA is elongated 5'-3'
Termination in eukaryotes
Not as well understood, mainly involving RNA poly 2
Sites of transcription/ translation in prokaryotes
Transcription and translation can occur almost simultaneously in the cytoplasm
Sites of transcription/ translation in eukaryotes
Transcription and mRNA processing occurs in the nucleus, translation occurs in the cytoplasm (ribosomes).
5' cap is only on transcripts generated by...
RNA polymerase II
5' cap consists of an additional (blank) at 5' end
guanine
When is the 5' cap added?
shortly after transcription initiation
What does the 5' cap do?
inhibits degradation by nucleases, promotes translation, and is necessary for export of mRNA
How does the poly(A) tail affect stability?
The poly(A) tail stabilizes the 5' cap, which must be removed before the mRNA molecule can be degraded from the 5' end.
are introns common in eukaryotic or prokaryotic genomes?
eukaryotic, found in nuclear, mitochondrial and chloroplast DNA
can functional RNA be obtained with introns included?
no, they need to be removed
what is needed for splicing in nuclear-derived pre-mRNA?
3 consensus sequences, 5' splice site (GU), 3' splice site (AG) and the branch point (adenine)
Alternative Splicing of mRNA
The same pre-mRNA is processed in diff ways, multiple 3' cleavage sites creates pre-mRNA of different lengths and the order of introns is maintained
Translation
synthesis of a protein with a specific sequence of amino acids based on the nucleotide sequence of an mRNA molecule
four major components of a translation system:
mRNA, ribosomes, tRNA and aminoacyl-tRNA synthetases
tRNA does what?
brings in amino acid as dictated by mRNA, smallest RNA molecules
aminoacyl-tRNA synthetases
An enzyme that joins each amino acid to the appropriate tRNA, can read language of both nucleic acids and proteins
What are the steps for translation?
1. binding of amino acids to tRNAs, initiation, elongation, and termination
What is needed for initiation to occur?
mRNA, small/large subunits of ribosome, three protein IFs (IF3,2 and 1), initiator tRNA with f-met attached and guanosine triphosphate for energy
What happens during initiation?
mRNA molecules bond to small unit of ribosome, initiator tRNA binds to mRNA and large ribosome joins
Where does initiation begin?
the consensus sequence upstream of the AUG start codon
Prokaryotes consensus sequence
shine delgarno, start codon downstream
eukaryotes consensus sequence
kozak sequence, start codon included
what recognizes consensus sequences?
small ribosomal subunits, place ribosome in the correct position
prokaryotic vs eukaryotic ribosomes
Eukaryotes - 80S (60s,40s)
Prokaryotes - 70S (50s,30s)
at the end of initiation...
the ribosome is assembled on the mRNA and the first tRNA is attached to the initiation codon
Elongation
the increase of the polypeptide chain by one amino acid
what dictates the type of tRNA molecules that will bind to the polypeptide chain?
the sequence of the next codon in the mRNA
what are the two binding sites in the ribosome?
P-site (P = peptide) and the A-site (amino acid); mRNA binds to the ribosome so that the first codon is in the P-site and the second codon is in the A-site
P site of ribosome
first site, bound to f-met
A site of ribosome
second site, receives second amino acid
once both P and A sites have bound tRNA the amino acids are linked by what?
a peptide bond (pepitidyl transferase)
P site releases its tRNA molecule which allows for what?
allows ribosome to move down the mRNA molecule, tRNA that was bound to the A site gets moved to the open P site which opens a site for the next tRNA molecule
Termination
elongation continues till the A binding site reaches a stop codon
why does a stop codon start termination?
stop codons do not specify an amino acid so tRNA does not bind at a stop codon which causes the finishes polypeptide chain to be released from the P site
what happens when the finished polypeptide chain/ protein is released from the P site?
the ribosome releases from the mRNA molecule
why is is good that mRNA is degraded within a few minutes or hours?
if they remained stable then the cell would not be able to quickly turn off the expression of a gene
operon organization
promoter and regulator gene which binds to operator
operator
part of an operon; DNA sequence between (at the border of) the promoter and structural genes
promoter
controls expression of structural genes
regulator gene
encodes the regulator protein, influences transcription
negative transcriptional control
Regulation of transcription by a repressor protein. When bound to the repressor-binding site, transcription is inhibited.
positive transcriptional control
Control of transcription by an activator protein. When the activator is bound to the activator-binding site, the level of transcription increases.
Inducible vs. Repressible operons
transcription is normally off with inducible and on with repressible
(-) inducible operons
transcription is normally off, repressor is normally bound to operator/promoter, turn on by changing shape of repressor with regulatory protein
(-) repressible operons
normally transcribing, repressor normally not bound to operator/promoter and must be turned off by activating repressor
(+) inducible operons
transcription normally off, activator is normally not bound to operator/promoter and substrate must activate
(+) repressible operons
normally transcribing, activator is normally bound to operator/promoter and the product of transcription de activates
The Lac Operon is an example of what kind of control?
Negative inducible meaning the repressor's shape must be changed by a regulator protein
When no lactose is present
repressor gene product binds to the lac operator, blocks RNA polymerase so there is no transcription
When lactose is present
allolactose binds to the repressor gene, preventing it from binding to the operator, RNA polymerase binds and there is transcription
Is glucose or lactose preferred by cells?
glucose, more efficient because no need to metabolize lactose
lacZ
Encodes B-galactosidase, which converts lactose into glucose and galactose for use in glycolysis
when glucose is present, what happens to the lac operon?
it is not induced by lactose and the CAP mechanism controls repression
CAP mechanism
Catabolite activating protein, example of positive control, must bind to promoter region
CAP with no glucose
binds to cyclic adenosine monophosphate (cAMP), low glucose=high cAMP which alters the structure of CAP and binds to the promoter
CAP in presence of glucose
cAMP levels not sufficient, high glucose=low cAMP, so the complex is not formed, promoter binding does not occur and lac operon is not transcribed
What type of control is exhibited by the Trp operon?
Negative repressible control meaning transcription is normally active and must be turned off by activating a repressor
low tryptophan levels
repressor can't bind to DNA operator allowing RNA polymerase to bind and transcription occurs, tryptophan results
Tryptophan present
tryptophan serves as a co-repressor and binds to the repressor which then binds to the DNA operator. RNA polymerase cannot bind therefore no transcription occurs
Other method of regulation for the Trp operon?
attenuation- to reduce in strength or weaken; 1+2 and 3+4 terminates transcription and 2+3 is antitermination
excess tryptophan and attenuation
regions 3+4 pair, a hairpin forms and stops transcription, known as a terminator structure
limited tryptophan and attenuation
regions 2+3 pair, an alternative hairpin is formed, transcription occurs and this is known as an antiterminator structure
eukaryotic vs prokaryotic gene regulation
regulation is more complex in eukaryotes due to more genetic information packaged as chromatin, site of transcription (nucleus) is separate from translation (cytoplasm), different genes used
mRNA half-life in prokaryotes vs eukaryotes
prokaryotes decay in minutes, eukaryotes take much longer
chromatin
Clusters of DNA, RNA, and proteins in the nucleus of a cell
what makes up the majority of chromatin proteins?
histone proteins which are many positively charged amino acids (lysine and arginine) that bond to (-) charged nucleotide phosphate groups
beads on a string" refer to what?
nucleosomes
chromatin remodeling
occurs prior to transcription, chromatin relaxes and opens in order to allow transcription to happen. Promoters are exposed when opened allowing enhancers and transcription factors to regulate transcription
transcriptional activator proteins
bind to specific DNA sequences and bring about higher levels of transcription by stimulating the assembly of the basal transcription apparatus at the start site
how do enhancers stimulate transcription at a distance?
they are time and tissue specific, can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur
what happens when a repressor binds near an activator site?
this prevents the activator from contacting the basal transcription apparatus resulting in no transcription
how do exonucleases degrade?
from the 5' or 3' end
how do endonucleases degrade?
by making an internal cut
process of ribonuclease degradation
3' end is degraded gradually till polyA binding proteins can no longer bind, the 5' cap becomes exposed and is removed
What is the basis of evolution?
genetic variation
mutations
refer to specific changes in DNA sequence, major source of genetic variation and can be bad or good
somatic mutation
cannot be transmitted to next generation, occurs in nonreproductive cells and goes through mitosis creating cloned mutant cells
gametic mutation
mutations passed to 1/2 of offspring, all cells of offspring will carry mutation, meiosis and sexual reproduction allow for this
is classification of mutations based on genotypes of phenotypes?
phenotype
base substitution
changes the base of a single DNA nucleotide/ codon; two types, transitions and transversions
base insertion
mutation which results in the addition of nucleotide pairs in a gene; can have a major effect on the resulting protein and can change many codons
base deletion
mutation which results in the loss of nucleotide pairs in a gene; can have a major effect on the resulting protein
transition vs transversion mutation
Transition- a purine is converted into a purine or pyrimidine is converted into a pyrimidine.
Transversion: a purine is converted into a pyrimidine (or vice versa)
frame shifts
Insertion or deletion of base-pair. Results are missense, nonsense and silent mutations
missense mutation
new codon codes for a different amino acid and there is a change in amino acid sequence
nonsense mutation
new codon is a stop codon and there is premature termination of translation
silent mutation
new codon encodes the same amino acid and there is no sequence change
forward vs reverse mutation
forward- changes the wild type into a mutant phenotype
reverse- restores the wild type gene and the phenotype
supressor mutation
genetic change that hides or surpasses the effect of another mutation, do not confuse with reverse mutation
consequences of mutations at the transcriptional level
disruption of TATA box and binding of transcriptional activator protein, mRNA processing disrupted at all steps
consequences of mutations at the translational level
creation of a stop codon (UGG to UGA) and interruption of ribosome binding
consequences of mutations during post-translational protein processing
the recognition site for modification may have been changed
How do mutations occur?
spontaneous or induced
spontaneous vs induced mutations
spontaneous: random change in the DNA arising from errors in replication
induced: results from exposure to known mutagens (happens more often)
wobble pairing
thymine on O.G template strand base-pairs with guanine through wobble which leads to an incorporated error, at the next round of replication the guanine nucleotide pairs with cytosine which leads to a transition mutation
strand slippage
either the newly synthesized (5'-3') strand loops out resulting in the addition of one nucleotide on the new strand or the template (3'-5')strand loops out resulting in the omission of one nucleotide on the new strand
unequal crossing over
If homologous chromosomes misalign during crossing over, one crossover product contains an insertion and the other has a deletion
base analog mutagen
induced mutation, can sub for purines or pyrimidines and leads to tautomeric shifts and transitions
alykylating agents
induced mutation, donates alkyl group to nucleotides forming analogs which cause transition mutations
intercalating agents
induced, cause frameshift mutations
ionizing radiation
highly reactive molecules cause single and double stranded breaks, singles are repairable but double can lead to chromosome rearrangements
UV radiation
primarily affects bacteria, causes pyrimidine-dimers that block replication and are lethal
Purpose of Ames test
use specific bacteria to evaluate the mutagenic properties of potential carcinogens utilizing 4 strains of salmonella which identify reverse mutations (his- to his+)
specific sensitivities of salmonella strains in an Ames test
one detects base pair deletions and three detect frameshift mutations
DNA repair mechanisms
Any of several processes by which enzymes repair DNA damage and avoid the accumulation of mutations
proofreading
a function of certain DNA polymerases, 3' to 5' exonuclease activity provides ability to "back up", recognizes improper hydrogen bonding and is specific for unpaired bases
mismatch repair
errors that remain after proofreading activate this process; removal of distorted segments and re synthesis with new bases.
template strand is methylated, daughter strand is not and the repair enzyme recognizes this mismatch, binds to unmethylated strand
base excision repair
recognizes/ replaces modified or incorrect bases, has minor effects and does not block DNA replication or transcription
5 steps of base excision repair
1. modified base is identified and excised by DNA glycosylase
2. AP endonuclease cuts the phosphodiester bond
3. deoxyribose removed
4. DNA polymerase fills gap (DNA poly 1 in prokaryotes and DNA poly beta in eukaryotes)
5. DNA ligase seals nick
direct DNA repair
does not replace like in a mismatch repair, returns damaged bases to their original state through enzymatic reactions
photoreactivation repair
Enzymes called photolyases use light energy to break the extra bonds in a pyrimidine dimer
Enables UV-damaged fungi to recover from exposure to sunlight
Humans do not have this type of repair
nucleotide excision repair
repairs lesions that distort the double helix, found in all organisms
transposition
the ability of some DNA elements to excise themselves and move to another position within the genome
Transposons (transposable elements)
mobile genetic elements that result in mutations, identified in all organisms, we will probably be tested on transposons in Maize
two forms most maize transposons exist in
autonomous elements and nonautonomous elements
autonomous elements
capable of transposition by themselves, known as an activator (Ac)
nonautonomous elements
only transposed if an autonomous element is also present, known as a dissociation element (Ds)
What type of element is the Ac-Ds system in maize?
autonomous element, controls activity of Ds elements which cannot transpose without an Ac element present
Ac-Ds system in maize
Activator (Ac)--> transposable element
Dissociation (Ds) --> no functional transposase.. requires AC to move around
goal of recombinant DNA technology
to combine DNA from two sources
restriction enzymes, types of cuts
endonucleases that are used to cut DNA strands at specific sites (usually palindromic sequences read the same forward and back); cuts can be staggered or blunt
staggered ends
produces single stranded cohesive sticky ends, ex: Hind III
blunt ends
(4/20 blaze it) produces blunt ends, ex: Pvu II
DNA molecules with the same recognition sites are cut with the same...have complimentary sticky strands
restriction enzyme, pair if fragments are mixed together
What is gel electrophoresis used for?
separate DNA fragments by size after splicing
Charge of DNA
Negative due to phosphate groups
during gel electrophoresis, (blank) fragments move quicker than (blank) fragments
small move quicker than larger
gene cloning
placing a fragment of DNA in a bacterial cell and allowing the cells to replicate producing identical copies of the DNA
What is the use of a vector?
origin of replication ensures replication inside host cell, selectable markers enable cells with the vector to be identified, and unique restriction sites allow it to be easily inserted into the host cell
vectors can be:
plasmid, cosmos, or artificial chromosomes
once a gene is placed inside a plasmid, it must be introduced into host usually by...?
transformation
LacZ gene as a plasmid
contains many restriction sites which allow for easy insertion, if foreign DNA is inserted, B-galac. is not produced and you can plate the bacteria to see if it contains a plasmid or not
what do white colonies mean?
they contain foreign DNA, the lacZ gene is not being expressed
what do blue colonies mean?
lacZ is functioning
PCR
polymerase chain reaction, most widely used tool in molecular biology
PCR uses...
heat stable DNA polymerases, like Tac polymerase
known primers that PCR requires
dNTPs, Mg2+,buffer and salt
PCR steps
1. Denaturation
2. Annealing
3. Extension
Denaturation
melting of two DNA strands by heating to 94 C, double to single stranded DNA
primer annealing
cool reaction to 50-70 C, mark region that is going to be amplified; the primers have a known sequence designed by the researcher
primer extension
heat up to 72 C using taq, new nucleotides are brought in
amplification is...
exponential
gene library
A collection of cloned DNA fragments, many clones are needed to cover an organisms genome
genomic library
contains all the DNA sequences found in the genome
cDNA library
A limited gene library using complementary DNA. The library includes only the genes that were transcribed into mRNA
Agrobacterium tumefaciens
a tumor-inducing (crown gall) bacteria in plants that has been altered by scientists to transfer traits (genes) from one plant to another; plant tumor makes compounds the bacterium needs
how to make the Ti plasmid easier to handle
genes needed for T-DNA can be moved onto a second plasmid which reduces the size of the Ti plasmid
how many plasmids are involved in agrobac. tume. transformation?
two, the helper Ti plasmid and the plasmid vector
Alt splicing vs multiple 3' splicing
Alt= same pre-mRNA processed in different ways
Multiple 3'= two or more possible 3' splice sites exist creating pre-mRNA of diff lengths
both just different versions of the same gene
What are the 3 consensus sequences needed for splicing?
5' splice site (GU), 3' splice site (AG) and the branch point
GAL4 in the absence of galactose
GAL80 blocks GAL4 from activating transcription
GAL4 in the presence of galactose
Galactose binds with GAL3 which changes GAL80's conformation, freeing up GAL4 to interact with the basal transcription and stimulate transcription