Lecture 7 Slides Flashcards
What does DNA polymerase require at chromosome ends
A primer
What is the main purpose of a telomere?
To stabilize chromosome ends
At what end of DNA does replication end? What is incomplete at end of replication?
End of replication of DNA is at 5’ end. At the end of replication, replication of lagging strand is incomplete.
Without telomeres, what would happen to 5’ end of DNA?
It would continue to shorten over successive rounds of DNA replication
Telomerase has homology to what?
Reverse transcriptase
How does telomerase work?
It synthesizes DNA strand (5’ to 3’) from RNA template that is part of the enzyme
What does DNA polymerase do after telomerase?
It elongates opposite strand
What is human telomere sequence?
T2AG3
Steps of telomerase function
- Telomerase binds to incomplete lagging strand at end of replication
- Binds to parental strand at 3’ and adds to it.
- Extends three end with RNA templates DNA synthesis (G strand)
- DNA polymerase completes lagging strand (C strand)
What is shelterin complex
Protects telomere DNA and recruits telomere at S phase
What does shelterin complex regulate
Formation of t-loop
Which shelterin complex proteins bind single strand DNA
TPP1 and POT1
Which shelterin complex proteins bind double strand DNA
RAP1, TIN2, TRF2, TRF1
What is the difference, at end of DNA replication,between parent and lagging strands?
3’ overhang
How is t loop formed
Overhang loops above parent strand and links into it
In what cells is telomere active
In germ cells and some stem cells
Where does telomere DNA gradually shorten? What does that do to its activity?
In somatic cells
Activity is low
What do humans with with mutation in one copy of telomerase gene display?
Premature aging
When do primary cultures of animal somatic cells cease to divide?
After ~50 cell cycles; telomerase shortens at each cycle
What is function of DNA surveillance system
Detects chromosome defects, inhibits cell cycle or promotes cell death
If telomerase is active in cells, do they proliferate
Yes, indefinitely
What allows transformed cell lines and cancer cells to divide indefinitely
Active telomerase
What enzyme is used to make rna from DNA?
Rna polymerase
Difference between ribose and deoxyribose
Second carbon in deoxyribose has H i/o OH
Difference between uracil and thymine
Uracil carbon double bond has no methylated carbons, but thymine has one methyl attached to carbon
Two types of dna dependent rna polymerases
Multi-subunit polymerases and single-subunit polymerases
Bacteria multisubunit polymerase
-one polymerase synthesizes all RNAs
alphaI, alphaII, beta, beta’, + sigma
-polymerase binds DNA via alpha subunits, sigma factors
-beta subunit has polymerase activity
-homology of all five bacterial proteins are found in archaea and eukaryotes
Archaea multisubunit polymerases
One polymerase; 11-13 subunits;
Closely related to eukaryotic polymerases
Eukaryotes multisubunit polymerases
Three polymerases; Pol I - 14 subunits; Pol II-12 subunits; Pol III - 17 subunits
Pol IV and pol V - roles in plant siRNA function
Single subunit polymerases
Bacteriophage T7 RNA polymerase
Mitochondrial RNA polymerases
Some chloroplast RNA polymerases
Where can one find RNA dependent RNA polymerases
In some viruses (like polio virus)
In eukaryotes, play a role in mi-RNA and si-RNA pathways
A-amantin sensitivity of RNA polymerase I. Genes transcribed
Insensitive
5.8S, 18S, rRNA genes
A-amanitin sensitivity of rna polymerase II? Genes transcribed.
Very sensitive
Protein coding genes, miRNA, siRNA genes
A-amanitin sensitivity of RNA polymerase III. Genes transcribed.
Intermediate sensitivity
TRNA genes, 5s rRNA genes, other small RNA genes
Which dna strand is used as template
Either. It depends on promoter orientation.
Can there be multiple rna polymerases transcribing
Yes
What controls rate of transcript accumulation
Rate of initiation
Steps in transcription cycle of RNA polymerase II
- Pre-initiation complex (PIC) formation
- Pol II recruitment
- Transition to initiating pol II
- Transition to elongating pol II
What happens during PIC formation?
Assembly of TFIID
TATA-box binding protein (TBP) + 13 other proteins at TATA box
What ahopens at pol II recruitment?
Assembly of additional general transcription factors and RNA pol II
Transition to initiating POl II
Helicase activity in TFIIH pries apart dna helix
Protein kinase activity in TFIIH phosphorylates residues in c terminal domain (CTD). CTD is unique to POL II largest subunit; repeated heptapeptide:
Tyr-Ser-pro-Ser-pro-Ser
Binds RNA processing factors
What happens during transition to elongate pol II
Pol II is released from initiation factors, elongation begins
What does preinitiation complex in vivo include
General transcription factors
Pol II
Mediator- a structurally flexible of about 25 proteins that links transcription factors (activators or repressors) to TNA pol II
Proteins for modifying chromatin structure
Steps in eukaryotes RNA processing
- DNA is in nucleus
- DNA has introns and exons
- rna is transcribed from transcription unit into primary rna transcript
- primary rna transcript gets 5’ capping, RNA splicing, 3’polyadenylation
- becomes mRNA, which is then exported from nucleus
- mRNA is then translated into protein outside of nucleus
Prokaryotic rna processing pathway
DNA is in cytosol ( no nucleus)
Transcribed directly to mRNA
Translated in cytosol
Processing enzymes carried by C-Terminal domain
Capping factors at 5’ end of mRNA
Splicing proteins
5’ cap
3’ end processing proteins
What makes up eukaryotic mRNA cap?
5’-to-5’ triphosphate bridge
7-methylguanosine
Difference between polycistronic and monocistronic mRNA?
Poly has coding for multiple proteins.
Mono has coding just for one protein
How is 5’ end cap added
Phosphatase takes phosphate
Guanyltransferase add guanosine to end
Methyl transferase adds methyl group to base
On so e caps, add methyl group to ribose
Purpose of intron splicing
Facilitates evolution of new proteins
Provides opportunity for regulation of gene expression
Alternative splicing allows numerous proteins from a single gene
Splicing step 1
- U1 snRNP forms base pairs with the 5’ splice junction
2. BBP (branch point binding protein) and U2AF (U2 auxiliary factor) recognize the branch point site
Splicing step 2
U2 snRNP displaces BBP and U2AF and forms base pairs with branch point site consensus sequence.
Splicing step three
The U4/U6.U5 “triple” snRNP enters reaction. U4 and U6 snRNAs are held firmly together by base pair interactions. Subsequently, rearrangements break apart the U4/U6 base pairs, allowing U6 to displace U1 at the 59 splice junction. This creates the active site that catalyzes the first phosphoryl-transferase reaction.
Splicing step four
Additional RNA-RNA arrangements create the active site for the second phosphoryl-transferase reaction, which then completes the splice
Processing at 3’ end of transcript
- Cleavage and poly-A signals encoded in DNA
- Cleavage stimulation factor (CSF) and cleavage and polyadenylation specificity factor (CPSF) are attached to RNA polymerase
- RNA is cleaved
- Poly-A polymerase enters. Rna polymerase eventually terminates
- Poly-Abonding proteins enter process
- Poly-A length is regulated
What is polyadenylation signal
AAUAAA
At what sequence is there cleavage?shat is added at cleavage?
CA. Add hydroxyl group.
Where does gene rich dna lie in nucleus
In euchromatic regions
FISH stands for
Fluorescein in situ hybridization
Where are alu sequences found with FISH
In gene rich dna
What does fluorophore link to in fish
Antibodies
Transcription factories
Contains 8-10 RNA POL II molecules
Nuclear export of pol II transcripts
“Export-ready DNA exits through nuclear pore complex into cytoplasm
- on RNA there is a nuclear export receptor, hnRNP proteins, and SR proteins
- 5’ end exits first
- in cytoplasm, there are initiation factors for protein synthesis (eIF4G and eIF4E)
- factors binds to 5’ end
- prepare for translation
