Midterm 2 Flashcards
Enzyme for DNa replication
DNA polymerase
What are the three steps of transcription?
Initiation, elongation, and termination
How does the DNA polymerase add a nucleotide? (Chemistry)
The 3’ oh attacks the alpha phosphate of an incoming dNTP
Role of the exonuclease
It has a high affinity for incorrect pairs and degrades the DNA from the 3’ end
Processive versus non-processive
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When does chromosome replication occur?
In the s phase of the cell cycle
End replication problem
Since DNA synthesis needs an RNA primer to initiate DNA strands, the ends of DNA will have a hard time replicating.
Telomeres
In eukaryotes, to fix the end replication problem, they have telomeres which are TG rich seq that doesn’t code for anything
Telomerase
A special DNA polymerase that only creates the telomeres. It has an RNA component and so it is a ribonucleoprotein and doesn’t need an template to add bases. It’s also like a reverse transcriptase because it uses the RNA template to make dna
What end does the telomerase act on?
It acts on the 3’ end
General causes of mutation
Environmental factors like chemicals and UV light, tautomerization, wrong base pairing, transposons, inaccuracy in replication
Transition mutation
When a purine is switched with another purine or pyrimidine with pyrimidine
Transversion mutation
Purine to pyrimidine switch
Point mutation
Mutations that alter a single nucleotide
Why are DNA micro satellites prone to mutation?
DNA microsatellites are long repeating seq like CACA and they are harder to replicate because slippage may occur. Therefore the repeat length may vary depending on the individual
Mutagen
A chemical that increases the rate of mutation
Deamination of cytosine
This is one of the most common mutations and turns cytosine into uracil
DNA depurination
This is when there is spontaneous hydrolysis of the N glycosyl linkage and it produces a deoxyribose without the base
Thymine dimer
When two thymine bases fuse to make a cyclobutane ring and this causes DNA polymerase to stop during replication if it reaches this point
How do gamma radiation and x-rays damage DNA?
They break the double strand which is really difficult for the cell to repair
Intercalating agents
They are able to slip between bases and cause errors in replication. They do this by causing additions, deletions, or even frame shifts
Base excision repair
DNA glycosylase removes the base first, and then AP endo and exo nuclease remove the backbone. The gap is then filled with the correct base by DNA polymerase.
Photoreactiviation
Reverses the formation of pyrimidine dimers from uv radiation by using the energy from light directly to break the dimer bond
Nucleotide excision repair
The DNA is scanned by a tetramer (UvrAB) for distortions and if a distortion is detected, UvrC will cleave a segment with the lesion out.
Methyltransferase
It reverses the methylation of guanine by taking the methyl group and putting on its own cysteine
What can mutations in the genes coding for nucleotide excision repair cause?
Can lead to UV light sensitivity
Double strand break repair
Usually repaired by non homologous end joining but during replication is is fixed by homologous recombination.
Non-homologous end joining
Ku70 and Ku80 bind to the broken ends and recruit DNA PKcs which then recruit Artemis, an exo and endonuclease that will process the broken ends. Then lipase is recruited to to seal the ends together.
Homologous recombination
The broken strand uses the original parent strand as a template and then the copied area is switched with the template so now the repaired strand has part of the template strand within it.
DNA cloning
Selective amplification of a particular gene or DNA segment
The five general steps of DNA cloning
1) cut DNA at specific location (restriction endonuclease)
2) select a cloning vector that can self replicate
3) join your DNA fragment with the vector
4) put this recombinant DNA into a host organism
5) select the cells that have the recombinant DNA
What do you need for a successful DNA vector?
You need an origin of replication, a selectable marker, and a region in which your DNA can be inserted
Plasmid
A circular double stranded DNA molecule with a size from 1 to 200 kb. They replicate separately from the host cells chromosomal DNA
How to insert your DNA into plasmid
Use same cleaving enzyme like EcoR1 to make sticky ends. Then allow your DNA fragments to anneal and use ligase to seal them together
How to get vector into host cell
Use transformation for bacterial cells and transfection in mammalian cells
BACs and YACs
They are used to clone large segments. If you clone these within the lacZ gene, you can tell if the insert is present or not.
How to tell if BAC or YAC has insert
If it does have the insert, the lacZ gene will be disrupted and won’t turn color in media with x-gal.
DNA library
1) DNA digested with restriction enzyme
2) cleaved DNA mixed with vector ligase
3) this creates a DNA library because each vector has different DNA fragment. You can then screen for your gene of interest
Genomic versus cDNA library
A genomic library represents the entire genome while a cDNA library represents only the genes expressed in the cell since the mRNA was copied into DNA
Reverse transcriptase
Converts RNA sequences into DNA. Needs a primer and either DNA or RNA template . You can make tissue specific cDNA libraries
RNA polymerase in prokaryotes
Has beta core
RNA polymerase in eukaryotes
There’s I, II and III but we focus mostly on RNAP II because it is involved in mRNA synthesis. The others make RNA for ribosomes and tRNA
How many metal ions are involved in RNA polymerase?
Two Mg2+ ions are involved but one comes with the incoming rNTP
What direction is RNA made in?
From 5’ to 3’
Template strand versus the coding strand
The template strand is the one the RNA will be made complementary to, while the coding strand is the one the RNA is identical to.
Antisense versus the sense strand
Antisense = template strand Sense = coding strand
What does upstream mean?
It means toward the 5’ end of a given sequence
RNA polymerase holoenzyme
Only in prokaryotes, consists of the RNA polymerase core (5 subunits) and the sigma factor
Job of sigma factor
It recognizes the promoter region and helps the RNA polymerase bind to the promoter region.
Promoter region (types)
The most common promoter region has a -10 and -35 element. A stronger promoter might have a UP element. The third type has no -35 but instead has an extended -10. The last type has a discriminator next to the -10 region.
What recognizes the UP element?
Alpha CTD (or the carboxyl terminal domain of the alpha subunit)
Steps within transcription initiation
Closed complex = binding of sigma factors to promoter region
Open complex = DNA begins to be unwound
Initial transcript = short RNAS are made and released repeatedly until elongation phase begins
How does the RNA polymerase transition to the open complex?
The sigma 1 factor moves out of the active site and the promoter melts
Steps of transcriptional elongation
RNA polymerase unwinds the DNA at the front, adds rNTPs, reanneals the DNA in the back, proofreads and dissociates the mRNA from the template
Hydrolytic editing*
The nuclease within the RNA polymerase cleaves off an entire segment of ribonucelotides
Pyrophosphorolytic editing
Look in book
Terminator
A sequence that triggers the RNA polymerase to dissociate from DNA and release the RNA
Rho independent terminator
A sequence rich in GC that will form a hairpin and cause a release of the polymerase without the need of a protein
Rho dependent terminator
When the protein factor rho is present, RNA synthesis will stop and every dissociates. These sequences have a rut site that rho binds to.
GTF’S
They are general transcription factors that are needed in eukaryotes to recognize and bind the promoter. Somewhat similar to the sigma factor in prokaryotes
The transcription factors needed for pol II
TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH
The main promoter for eukaryotes
TATA box
What binds to the TATA box first?
TBP and TFIID bind first. They then recruit other GTF’s and pol II which forms the ore initiation complex
Steps for initiation in eukaryotes
Closed complex = melting of the promoter by TFIIH (needs ATP)
Open complex = phosphorylation of the polymerase tail by TFIIH which also needs ATP
Promoter escape = release from promoter and most general transcription factors
What is the last step needed before elongation occurs?
The mediator complex connects the activator sequence to the initiation complex. Chromatin remodeler and HAT are also needed
What happens in eukaryotic elongation?
The transcription initiation factors and mediator complex are shed and new proteins are recruited. Different phosphorylation of the tail exchanges initiation factors for the new proteins
What phosphorylation signals the capping enzyme in the promoter escape phase?
Phosphorylation of serine 5
What phosphorylation signals the splicing machinery?
Serine 2 phosphorylation
5’ cap
A modified guanine is added to the 5’ end of the mRNA. The beta phosphate of the chain attacks the alpha phosphate and creates a 5’ to 5’ linkage. This guanine is then methylated (modified). All eukaryotic mRNAs have a cap
Polyadenylation
Poly A tail is added to the 3’ end. CstF and CPSF bind to the tail, and then to the RNA when signal sequence is transcribed. CstF leaves after it cleaves the RNA and PAP is recruited. PAP adds about 200 Adenines to the RNA without a template and finally polyA binding proteins bind to the tail
Torpedo model
After the first RNA strand is cleaved off to make the polyA tail, the uncapped RNA is degraded by RNase and this destabilizes the RNA polymerase and causes termination.
Allosteric model
Once the polyA signal is passed, the RNA polymerase becomes less processive because of a conformational change
Function of polyA tail and 5’ cap
They stabilizes the mRNA, allow it to be transported out of the nucleus, and help with translation initiation
Gene expression is prokaryotes versus eukaryotes
In prokaryotes, transcription and translation can occur simultaneously. While in eukaryotes, there is transcription, then RNA processing, then transport, and finally translation.
Exon
Sequences that contain the genetic information and remain present within the final RNA product
Introns
Intervening sequences that are removed by RNA splicing
hnRNA
Heteronuclear RNA. RNA of many different sizes
Pulse chase experiment of RNA
In the pulse, RNA was labeled with 32P for 30 minutes. The RNA was then separated by rate zonal centrifugation. In the chase, they stopped transcription and allowed the cells to grow for 3 hours. They concluded that mRNA is derived from hnRNA.
Signals for RNA splicing
There’s the 5’ splice site, 3’ splice site and the branch site. They occur at exon/intron boundaries in most genes
The splicing reactions
It includes two transesterification reactions.
1) 5’ splice site is cleaved
2) the branching point OH attacks the 5’ end phosphate and forms a loop (lariat) 5’ to 2’ bond
3) the 3’ end is cleaved
4) the 5’ exon attacks the 3’ exon and they join together
What is the spliceosome made of?
Made of SnRNPs (small nuclear ribonucleoprotein particles) U1,2,4,5,6 which catalyze the reactions. They are ribozymes!
Which SnRNP recognizes the 5’ splicing site?
Usually U1
The steps of the spliceosome mediated splicing reaction
E complex, a complex, b complex, and finally the c complex
What happens in the E complex of splicing reactions?
U1 recognizes the 5’ binding site, BBP binds to the branching point, and U2AF binds to the py tract and the 3’ splice site
What happens in the A complex of splicing?
U2 comes in and replaces BBP
B complex of the splicing reactions
U4, U5 and U6 bind to the intron and form a complex with the others. U1 leaves and U6 takes its place at the 5’ splice site. U4 is then released and this allows U6 to interact with U2
C complex of RNA splicing
U6 and U2 catalyze the first transesterification reaction and then U5 helps with the second one.
A rare form of RNA splicing
Some rare RNA have the ability to splice themselves. They can do this by folding into a conformation that catalyzes it’s own release from the surrounding exons
Alternative RNA splicing
Occurs in 95% of human genes with introns. Allows multiple proteins to be made from one gene. It is often cell type specific.
Introns early model
Originally prokaryotes had introns too but lost them over time in order to replicate more efficiently.
Introns late model
Introns were inserted into genes that originally did not have introns
Exon shuffling
Allows the creation if new genes through duplication and recombination. It is one reason why introns have been kept in our genome.
What do you need for translation? (Key components)
mRNAs, tRNAs, and the ribosome
Three stages of translation
Initiation, elongation, and termination. Same as transcription!
How many codons are there?
There are 61 codons coding for 20 amino acids plus 3 stop codons for a total of 64 codons.
How many possible reading frames does mRNA have?
3 possible reading frames
Open reading frame
Starts with a start codon and ends with a stop codon
Which direction are codons read?
5’ to 3’
Silent mutation
When a nucleotide is switched but it still codes for the same amino acid
Missense mutation
When a nucleotide is switched and codes for a completely different amino acid
Nonsense mutation
When a nucleotide is switched and the new codon is now a stop codon
Which end of the tRNA holds the amino acid?
The 3’ end. It usually ends with CCA
Uncharged versus charged tRNA
Uncharged doesn’t have an amino acid and charged does
Secondary and tertiary structure of a tRNA
Secondary is like a cloverleaf with anticodon loop, variable loop, pseudouridine loop, d loop and acceptor arm. The tertiary structure looks like an L with the acceptor arm on one end and the anticodon loop on the other end
What enzyme charges the tRNAs and how does it do it?
Aminoacyl-tRNA synthetase requires ATP in order to add the amino acid to the tRNA. (20 different synthetases)
Adenylation
The carboxylic acid of the amino acid attacks the alpha phosphate of the adenine to make an adenylylated amino acid. This process requires ATP
How does tRNA charging occur? (Chemical process)
The adenylylated amino acid is attacked by the 3’ OH of the tRNA. This process releases the AMP.
Size of ribosome subunits in prokaryotes
The large subunit is 50S and the small subunit is 30S. This adds to 70S
Ribosome subunit size in eukaryotes
The large subunit is 60S and the small subunit is 40S to make a total of 80S
The ribosome cycle
Initiation, elongation, and termination
Polysomes
When multiple ribosomes can translate a single mRNA molecule simultaneously
Function of the small ribosome subunit
It mediates the interaction between the codons and tRNA anticodons
Function of the large ribosome subunit
Catalyzes the peptide bond formation, and has binding sites for G proteins that assist in initiation, elongation, and termination
Peptidyl transferase reaction
The reaction that adds the next amino acid to the chain (creating a peptide bond)
What direction are polypeptides synthesized?
From the n terminus to the c terminus
The structure of a ribosome
Has an a site or aminoacyl site, the p site (peptidyl) and finally the e site (exit)
Prokaryotic translation initiation
The 16s rRNA be pairs with the ribosome binding site (RBS) and this positions the start codon with the p site of the ribosome
Polycistronic
When mRNA contains multiple open reading frames
Initiation in prokaryotic translation
The small subunit binds to the start codon and then the initiator tRNA binds to the AUG and has a special methionine. The large subunit then comes in to form the complete ribosome
Function of IF3, IF2, and IF1
IF3 binds the e site and doesn’t allow the 50S to bind. IF1 blocks aminoacyl tRNA from binding to the a site. IF2 is a gtpase that interacts with IF1 and the special methionine tRNA. This leaves only the p site open for the initiator tRNA
Monocistronic
MRNAs with only one open reading frame
Why are eukaryotic mRNAs held in circles? And how?
This is so the ribosome can easily reassociate to make more protein. It is held in a circle by the binding of elF4A to the poly A tail
Release factors for translation in prokaryotes
The class 1 RFs release the peptide chain from the last tRNA. Class 2 RFs release the class 1 factors.
RRF
Ribosome recycling factor helps release the tRNAs and dissociate the ribosome subunits
