Type I Topoisomerases
cleave one strand of DNA
Type I Topoisomerases
change the linking number (Lk) by 1 or a multiple of 1 (monomeric)
Type II topoisomerases
cleave both DNA strands
Type II topoisomerases
couples energy from ATP hydrolysis to the introduction of negative supercoils
Type II topoisomerases
change the linking number (Lk) by a multiple of 2 (dimeric)
Type I and II topoisomerases
regulate DNA supercoiling
adds
DNA polymerase ___to existing DNA; it cannot start a new nucleotide strand.
okazaki fragments
The lagging strand is synthesized in pieces called
helicases
use energy from ATP hydrolysis to unwind nucleic acid and duplexes
DNA polymerases
may proofread newly synthesized DNA. Also contain an active site that can hydrolyze nucleotides at the 3' end if mismatched nucleotides are detected.
DNA ligases
join discontinuous DNA segments. During DNA replication, this continuous lagging strand.
Ribonucleases
degrade RNA, excising the RNA primer during DNA replication
DNA Polymerases
extend preeexisting deoxynucleotide chains
Primases
synthesize short RNA segments, which can then be extended during DNA replication.
telomerase
-extends linear chromosomes by adding nucleotides
- has reverse transcriptase activity
-contain an RNA component that serves as a template for a DNA sequence
- repeating sequence is TTAGGG
euchromatin
the less condensed form of chromatin
heterochromatin
the inactive form of chromatin
nucleosome
If DNA structure is described as "beads-on-a-string" a "bead" is a
heterochromatin
the more darkly-staining form of chromatin is
DNA molecule
If DNA structure is described as "beads-on-a-string" a "string" is the
nucleosome
A DNA protein complex
histone
A core composed of eight___proteins interacts with DNA through hydrogen bonding and ionic bonds
nucleosome
DNA winds around a core of eight histone proteins to form a
heating
The first step in PCR is to separate the DNA strands by
cooled
After the strands separate, the sample is ___, enabling the primers to bind to the DNA strands
DNA polymerase
After the primers bind to the DNA,___ lengthens the new DNA strands using the deoxynucleotides in the mixture
EcoRV
The sequence GAAGATATCCTCGC is cut by the restriction enzyme
Hind III or EcoRV
The sequence AAAGCTTGTCATATC can be cut by either ___ or ___
Prokaryotic promoter
-recognized by the o factor of RNA polymerase
-includes two conserved sequences at -35 and -10
Eukaryotic promoter
-require multiple proteins called general transcription factors
-may include a TATA box (not the Prbnow box) upstream of the transcription start site
Both eukarytoic and prokaryotic promoter
- may include sequences downstream from the transcription start site
-contains highly conserved DNA sequences
enhancer
interacts with proteins to increase the transcription of a gene
operator
often near a promoter, may interact with proteins to suppress transcription
activator
binds to a regulatory site on DNA, increasing the gene transcription rate
repressor
binds to a regulatory site on DNA, decreasing the gene transcription rate
DNA
Enhancer and operator are part of
Activators and Repressors
___ and __ are regulatory proteins that bind to an enhancers and operators.
Constitutive gene expression
Gene is always expressed at the same rate
Housekeeping genes
Genes with products that are always needed in the cell. Not always transcribed at the same rate
Regulated gene expression
the concentration of gene and products rise and fall depending on the conditions and the resulting molecular signals
Induction
increasing the gene expression of a gene
repression
decreasing the expressions of a gene
Increase in gene products
-The l gene is disabled. Because the I gene codes for the repressor protein, no repressor is made if the I gene is not functional. If the repressor is not bound to DNA, transcription occurs.
-lactose is present in the cell binds to the repressor, inactiva
Decrease in gene products
-Allolactose is not present in the cell.
-The Lac repressor remains bound to the operator
protein
Exons code for
Noncoding introns
___ ___ are removed from the RNA before it exits the nucleus.
prokaryotic 30S
IF-3, 30S ribosomal subunit, IF-1, and mRNA are components of the ___ ____ initiation complex that is required for protein synthesis.
Initiation
-kinetic proofreading occurs
-the first AUG codon is recognized, lacks the Shine-Dalgarno sequence
-in E.coli, mRNA binds to the 30S ribosomal subunit
Elongation
-peptidyl transferase catalyzes peptide bond formation
-an aminoacyl-tRNA enters the A site
-peptidyl transferase transfers the peptidyl group to water
-can cause the EF-Tu-aminoacyl-tRNA complex to leave the ribosome without bonding, aka kinetic proofrea
Termination
-in E.coli, an initiation factor block the A site
P
The ___ site binds the tRNA molecule that is attached to the growing peptide chain.
A
The site that binds the aminoacyl-tRNA is the ___ site
E
The ___ site may be bound to deacylated (uncharged) tRNA
P
The site that binds the peptidyl-tRNA is the ___ site
EF-Tu
Aminoacyl-tRNAs are ferried to the ribosome by
False
The transpeptidation reaction proceeds via ATP hydrolysis. True or False
rho
The transcription of genes in E.coli that lack a termination sequence require ___ factor to terminate transcription.
phosphorylation
During elongation, RNA polymerase is modified by___
To the alpha-carboxyl group of the amino acid
In the two step process of charging tRNA's, where is the AMP group initially added?
False
The acceptor stem is not crucial for an aminoacyl-tRNA synthetase-tRNA interaction. True or False
True
In eukaryotic promoters, many of the elements required to initiate transcription have variable positions and orientations relative to the transcribed sequences. True/False
sigma
Once RNA synthesis has been initiated in E.coli, the ___ subunit( or factor) dissociates from the core enzyme.
True
All cellular RNA's are transcribed from DNA templates. True/False
True
RNA polymerase synthesize RNA in the 5'-3' direction. T/F
False
RNA polymerase synthesize RNA that has the complementary sequence to the coding strand (with Us instead of Ts). T/F
True
RNA polymerase travels along the template strand in the 3' to 5' direction.
True
RNA polymerase is recruited to the promoter by general transcription factors.
Smaller fragments
Which size of DNA fragments will be at the bottom of an agarose gel?
True
DNA ligase is not essential in PCR. T/F
True
Dideoxy DNA sequencing is also called chain termination method. T/F
Negative supercoils
Supercoils in right-handed direction
-winding less frequent
Positive supercoils
supercoils in left handed direction
-winding more frequent
5' to 3'
DNA polymerase synthesizes __ to __
irreversible
DNA polymerase is ____ due to hydrolysis of PPi
meet
Replication terminates where two replication forks ____
circular, linear
Bacterial chromosomes are ___ while eukaryotic chromosomes are ____
chromosome shortening
Implication of Problem: Each round of DNA replication would lead to ___ ____
5' to 3'
Telomerase synthesis proceeds in __ to __ directioon
Template
Telomerase uses RNA molecule as ___
active site
The DNA polymerase is shaped like a hand. The palm is cleft where DNA binds and contains ____ ___
proofreader
3' to 5' exonuclease activity acts as ___
DNA
SSB (single-strand binding protein) concentration will be higher than that of ___ polymerase
MutS
Bacterial ___ protein binds mis-matched pair cause DNA to bend.
endonuclease
Mismatch repair: induces an ____ to cleave the strand with the wrong nucleotide.
oxidative
Damage by ROS are by-products of ___ metabolism
Base excision repair
-Removal and replacement of modified bases
-DNA glycosylase binds DNA and removes damaged base
-AP endonuclease nicks the backbone
-AP: apurinic/apyrimidinic
-DNA polymerase fills in the gap; ligase seals the nick
UV Damage repair
-Repair for thymine dimers
-Segment containing dimer +30 nt cut out
-Filled in by DNA polymerase; nick sealed by ligase
Nonhomologous end joining
-Ku undergoes conformation change & recruits nuclease
-Nuclease trims residues from end
-Polymerase recruited that utilizes template-independent polymerization
-Inherently mutagenic
-Permanent break in DNA
Oxidation of nucleotide bases
What type of DNA damage occurs due to reactive oxygen species?
Initiation
Bind polymerizing machine, first monomer to template. ( DNA polymerase, RNA polymerase, ribosome)
Elongation
Read template, add next monomer ( DNA, RNA, Protein)
Termination
Release machine and completed product
replicate
-To duplicate= to make an exact copy
-making an exact copy of the DNA
Transcribe
-to make a copy
-making a copy of the coding strand of DNA in the form of RNA
-Different "handwriting" (RNA vs. DNA) but same language (nucleic acid)
Translate
-To render in another language
-Converting the language of nucleic acid into the language of amino acids (proteins)
B-type
RNA does not form __ __ double helix
amino acids
mRNA trancripts --> sequence of __ __
Protein coding (DNA->RNA->protein)
Noncoding RNA's (DNA-> RNA)
Name the two types of genes
eight
A typical gene consists of ___ exons.
80%
What percent of the human genome may undergo transcription to produce noncoding RNAs ?
-No 3' OH on ddNTP to add onto
In the dideoxy DNA sequencing, why would the reaction stop?
Different fluorescent labels.
How do you know which ddNTP is which?
PCR
-These cycles are repeated 25-35 times
-Primer in one cycle becomes template in next
-Amplify amount of DNA
-Solution repeatedly heated and cooled
-Need thermostable DNA polymerase ( Taq and Pfu)
Only PCR requires a thermostable DNA polymerase
How does PCR differ from dideoxy DNA sequencing?
vector or plasmid
The carrier DNA
Recombinant DNA technology
-2 separate DNA molecules are recombined to form a new molecule
-Both DNA molecules digested with the same enzymes
-Both have the same sticky ends
-The digested molecules are recombined; DNA ligase seals the nick in the backbone
Cloning Vectors
-Plasmids that are autonomously replicated (seperate from chromosome and has its own origin of replication)
-Usually carry nonessential genes
-Usually carry selectable marker ( most often anitbiotic resistance gene)
-Often have multiple cloning site(multi
-Confers antibiotic resistance
-Grow cells in presence of antibiotic
-Only cells that have the plasmid and can express the resistance gene will survive
What is the purpose of selectable marker?
Enhancer
-Range from 50-1800 bp in length
-May be up to 120 kb upstream or downstream of promoter
-Bound by activators
-Mediators protein complex links activator to general TFs at promoter
-Requires looping of DNA
Intervening
Remember nucleosomes shorten length of ____ DNA
Silencers
-Bound by repressor proteins
-May also be connected to transcription machinery via Mediator
Mediator
-Complex of up to 80 proteins
- Different combinations may produce different results
-Recognize different activators/repressors
-Not a DNA-biding complex
Operon
-Multiple genes transcribed from one promoter (operator region)
-All genes transcribed together
Lac Z
The ___ gene encodes beta-galactosidase. Hydrolzes lactose to galactose + glucose
Lac Y
The __ gene encodes lactose permease. Lactose transporter
Lac A
The ___ gene encodes thiogalactoside transacetylase.
LacI
Repressor protein ___ blocks transcription.
RNA polymerase
-Binds daDNA, reads sequence
-reads 3' to 5'
-sythesizes RNA to 5' to 3'
-Does not need primer to being synthesis
-Only reads one strand ( template or antisense strand); synthesizes complementary strand ( coding or sense strand)
-Substrates are rNTPs
-Tho
less, mRNA, transcription
The double helix of DNA-RNA is wider and flater, therefore it is ___ stable and more easily dissociated. This type of hybrid is essential in the process of synthesizing ___ aka____
Rudder
___ protein loop may help separate RNA from DNA so that RNA exits.
bridge
Helix near the active site (____) oscillates between straight and bent conformation.
Transcription in Nucleus
-Processed before transport to cytosol for translation
-translation occurs in the cytosol
5' cap
-Protects mRNA from exonuclease digestion
-Phosphatase removes terminal phosphatase adds
-Guanylytransferase adds GMP from GTP
-Methyltransferase add methyl group
3' tail
-Sequence in transcript signal for cleavage
-Poly (A) polymerase generates a polyadenylate tail
-Poly (A) binding protein binds to help protect this end from nuclease digestion
exons
Eukaryotic genes that contain expressed regions
introns
eukarytoic genes that contain intervening regions
Splicing
Cut out the introns and connect the exons
Forms lariat intermediate
2' OH of branch point A residue attacks the phosphate at the 5' end of intron
Intron released
Free 3' OH now attacks 5' phosphate of 2nd exon
Active site Mg 2+ ion essential
-Makes OH more nucleophilic
-Stabilizes leaving group phosphate
-Differential splicing: one gene-multiple products
What are the benefits of splicing?
poly (A) tail
The half-life of mRNA depends on how rapidly the ___ ___ is shortened
Adenine and guanine
Name the purines
uracil, thymine, cytosine
Name the pyrimidines
A-T, C-G
Watson crick base pairs
major
B-DNA has a major and minor groove and the DNA-binding proteins bind in ___ groove
right, negatively
DNA is __ handed helix and is ___ supercoiled
Type IA
Which topoisomerase nicks the DNA and passes the strand through?
Type IB
Which topoisomerase nicks the DNA and allows one strand to rotate one turn?
Type II
Which topoisomerase directly alters number of writhes by passing dsDNA segment through another?
True
T/F: Neither glutamate nor glycine are needed for pyrimidine synthesis.
Mg2+/Asp side chain component of DNA polymerase coordinates phosphate group of nucleotides to position so 2' carbon would position in hydrophobic pocket. This binding site allows the polymerase to discriminate against ribonucleotides, which bear a OH grou
Why won't rNTPs bind?
Rho-independent and dependent
the two mechanisms for transcription termination in prokaryotes
As a means of protecting itself, the ssDNA portion inserts itself into the dsDNA portion
why the telomeres form the T-loop. What is its purpose?
shelterin
____ would determine how many of the telomere lengths are added.
it is a structural motif -- the way the protein folds. The intent is to have a small segment of the protein available to contact the major groove of the DNA and confirm the sequence -- by contacting the sequence of bases. The structure is simply two small
explain the helix-turn-helix motif?
We tend to have more repetitive sequences -- contributing to a larger DNA molecule overall. It's also true that the individual genes are larger -- because we have introns that will be spliced out and prokaryotes do not.
Why is the human genome larger than bacterial?
This means there are 3 possible reading frames for each strand of DNA.
How do we know how many open reading frames are possible in a strand?
RNA Polymerase I --> Makes rRNA
RNA Polymerase II --> Makes mRNA
RNA Polymerase III --> makes tRNA and other RNAs.
Know that there are multiple polymerases and which eukaryotic RNA polymerases make which type of RNA
DnaK
-clamps down on protein then releases it to allow it to fold
- binds to hydrophobic patches
Lys and Arg
Which AA residues would most likely be found in ribsomal proteins in large amounts?
Proofreading
Hydrolysis of GTP by EF-Tu is thermodynamically required for
P site
To which site on the 30S subunit does the initiator tRNA bind?
UUC
A tRNA carrying a 5' t 3' anticodon sequence of GAA would pair with a 5' t3' sequence of __ in the mRNA
to the alpha carboxyl group of the AA
In the two step process of charging tRNAs, where is the AMP group initially added?
irreversible
Aminoacyl-tRNA (requires ATP) synthetase reaction has subsequent hydrolysis of PPi makes this
translation
The process of translating the language of RNA into the language of protein
mRNA
contains the genetic code to specify the AA sequence of the protein
tRNA
carries the amino acids and reads the code
rRNA
part of the machinary that makes the protein product
specific, amino acid
Enzymes add amino acid to tRNA must be ___ and correct ___ ___