Lecture 7- Eukaryotic Transcription Flashcards
How many RNA Polymerase are there and name them?
3; RNA Polymerase I,II, III
Do RNA Polymerase share the same subunits?
They share some subunits
What is the fx. of RNA Polymerase I?
Synthesize precursors of ribosomal RNA
What is the fx. of RNA Polymerase II?
Synthesizes mRNA precursors. It recognizes promotors of varying sequences through assoc. proteins.
What is the Fx. of RNA Polymerase III?
Synthesizes precursors of ribosomal RNA, tRNA and other small RNA’s
How many subunits are on RNA Polymerase II?
12 subunits
What’s the difference between the subunits of Prokaryotic and Eukaryotic RNA Polymerase?
RBP1 subunit in Eukaryotic RNA Polymerase II has a long tail.
What sequence is on the strand that’s important for regulation?
YSPTSPS
What Amino Acid might be found on RBP1?
Prolines; loop typed structure
True or False. RBP1 forms a loop structure?
True
Fx. of Pol II
Catalyzes RNA Synthesis
Fx. TBP
Recognizes TATA Box
What is the TATA Binding Protein?
TBP
Fx. of TFIIA
Stabilizes binding of TFIIB and TBP to the promoter
Fx. of TFIIB
Binds to TBP; recruits Pol-II-TFIIF complex
Fx. of TFIIE
Recruits TFIIH; has ATPase and helicase activities
Fx. of TFIIF
Binds tightly to Pol II; binds to TFIIB and prevents binding of Pol II to nonspecific DNA sequences
Fx. of TFIIH
Unwinds DNA at promoter; phosphorylates Pol II whine the CTD; recruits nucleotide -excision repair proteins
Where is the initiation region?
+1
What are 4 steps of Transcription?
Assembly
Initiation
Elongation
Termination
What happens during assembly? what happens immediately after
TATA binding protein binds to the TATA Box
TFIIB, TFIIF-RNA Polymerase II, TFIIE, TFIIH.
What is the job of TFIIF after Assembly?
guiding RNA Pol II to the correct DNA sequence
What happens during assembly?
TBP binds to TATA box
TFIIB, TFIIF, TFIIE and TFIIH are all recruited to site.
TFIIF “Chaperone” guides RNA Pol II to correct DNA sequence.
TFIIH binding completes closed complex and TFIIH starts to unwind the DNA to create an open complex.
What causes initiation?
TFIIH phosphorylates RNA Pol II numerous times in its terminal domain, causing a structural change.
TFIIE and TFIIH are released as RNA Pol II synthesizes the first 60-70 nucleotides and starts elongating.
What happens during elongation?
TFIIF remains assoc. with RNA Pol II throughout Elongation. Additional elongation facts bind to the complex to enhance activity.
What causes termination?
Dephosphorylation of RNA Pol II,
True or False.
Prokaryotes can have transcription/translation simultaneously?
True
What are introns
Portions of non-coding sequences on DNA
What happens to introns?
They have to be spliced out.
Polycistronic mRNA Exists in ____.
Found in Prokaryotes
Why are polycistronic mRNA not common in Eukaryotes?
You need a 5’ cap to define the start site for protein biosynthesis.
What does the 5’ cap consist of?
7 methylguanosine
Wha happens to the 3’ end?
has to be cleaved and add a Poly A tail
What is the prokaryotic version of introns?
transpositrons
Introns get spliced after they leave RNA Pol II
True; happens in real-time
If I see a 5’-5’ Triphosphate Linkage what might that indicate?
5’ end cap of a mRNA
What groups are added to the first bases on a mRNA strand that might denote a 5’ end?
Methyl Groups placed on C2
5’ Cap protects mRNA from ______ .
Ribonuclease
5’ Cap might enhance binding assoc. with ____.
Ribosome
Cap is methylated at ____. before of after it’s added mRNA
N7; after
Cap comes from which nucleotide?
GTP
At what point is the cap added in reference to transcription?
Early in transcription
Enzymes that make up the cap are tethered to polymerase ______.
CTD
Which nucleotides of mRNA are methyl groups added to?
1st/2nd
What are the (4) enzymes in Cap synthesizing complex?
Phosphohydrolase
Guanylytranserase
Guanine-7-Methyltransferase
2-O’ Methyltransferase
What is the fx. of Phosphohydrolase?
Attacking gamma phosphorous position with water.
Gamma-P has been kicked off.
What is the fx. of Guanylyltransferase?
Beta O- is nucleophile; hits alpha-P atom in GTP (electrophile). It kicks off beta/gamma from GTP.
This moves G-Alpha to make a 5’-5’ Triphosphate
When is the cap Methylated?
Cap is methylated on N-7 and after it is added to mRNA;
after attack on GTP
Guanine 7-Methyltransferase
5’ cap comes from___
Guanylate
What adds the Methyl group to capping?
Guanine-7- Methyltransferase
SAM is the source of what?
Methyl group
SAM gives his methyl to____.
5’ end of mRNA during capping. Putting it on 7 methyl guanosine
What is the fx. of 2’ O Methyltransferase?
It determines where it wants to add the methyl groups. Either 1’ OH or 2’OH
The methyl groups are derived from ______.
S-adenosylmethionine
What happens after 7-methylguanosine is formed?
Capped 5’ end is released from the capping enzymes and bound to the cap-binding complex.
What is Actinomycin D
Deforms the DNA and prevents movement of RNA Polymerase.
Rifampicin
binds to beta subunit of bacterial RNA poly. and prevents extension past the promoter.
Alpha-Amanitin
Blocks RNA Pol II, but not bacterial RNA polymerase. Prevents predators from destroying the mushroom population.
Poly A tails are on what end?
3’ end
Poly A Tail cause degradation of bacterial
True
Tail length transcribes life or RNA
True
Bacterial love Poly A Tails.
False; they degrade poly a tail
What is the recognition seq. to lay down poly A tail?
AAUAAA
How many residues make up Poly A tails?
80-250
True or false
Poly A tails in Eukaryotic contribute to mRNA enzymatic destruction
False; Poly A Tails help protect mRNA from enzymatic destruction
What is the difference in Poly A tails destruction in Eukaryotic vs Prokaryotic mRNA?
Prokaryotic: stimulate decay
Eukaryotic: prevent degradation
What generates the free 3’ hydroxyl group that defines the end of mRNA?
Cleavage
What happens to RNA Polymerase when Enzyme Complex reccog. binds to transcript what does RNA Pol II do?
RNA Polymerase leaves
Endonuclease
It cuts the transcript
Poly A Polymerase
Put out 80-250 AA residues on the tripped end
Intron
Non-Coding
Exon
Coding
Which are longer? Exon or Intron
Intron
How many types of Introns are there?
4
Group 1/2 self-splice
True
In what major way do Group 1/2 differ?
Mechanism
Majority of eukaryotic introns are?
Spliceosomal Introns
Group 1 Introns use ____ to initiate splicing process
Guanylate
During Group 1 Intron nucleotide attacks, which end is attacked first and then what happens?
3’ end; it generates a 3’ OH that will then attack at the 5’ end of the second exon.
What happens after the introns after they are cut out?
Released/Degraded
Do Group 1 Introns appear to have consensus splice sites?
No
Group 1 Introns do NOT appear to have _______.
consensus splice sites
Explain process of Group I Introns
3’ OH of Guanylate (Nucleophile) attacks 5’ (Electrophile) Phosphoryl.
Kick off exon as a leaving group. Frees up 3’ OH Exon.
Guanylate is covalently bound to intron.
Now, free 3 OH’ (Nucleophile) attacks P on 3’ Intron.
Now (2) exon are fused
Adenosine is used in which intron group of splicing?
Group II Introns
Which Intron group produces a lariat structure?
Group II Introns
Which intron group uses a 2’ oH
Group II Introns
Does Group II Introns have consensus splice sites?
Yes
What does the Lariat Structure have?
3 Phosphodiester Bonds
Describe the process of Group II Intron
2’ OH of Adenosine of Intron (Nucleophile) attacks the 5’ splice site to form a lariat structure.
2’ OH (Nucleophile), Attacks P atom in phosphodiester linkage that bridges exon and intron. It will sever the bond.
Electrons now kick off exon. Now we have a free 3’ OH
Now we form a Lariat Structure.
3’ OH (Nucleophile) hits phosphorous bride between intron and exon.
Kicks off intron as leaving group. Now we have a phosphodiester bond bt. exon.
What nucleases are o the 5” and 3’ end during Group II Introns?
5’ GU
3’ AG
Consensus Splice Site in
Group II
GU-AG
What is a notable feature about the Lariat Structure
3 Phosphodiester Bonds
In what ways are the structures directed in the Lariat Structure?
(2) runs 5’ - 3’
(1) runs 2’ to 5’
How many RNA complexes are involved in spliceosomal introns?
5
True or False
Group II Introns are self splicing?
True
