Week 8 RNA Transcription, Processing and Decay

Question 1

What are the different classes of RNA in a cell?

Answer 1

mRNA: the intermediary that passes information from DNA to protein

Functional RNA (ncRNA) - the final functional product

Question 2

What type of functional RNA regulates protein synthesis?

Answer 2

tRNA and rRNA

Question 3

Template vs Non template DNA strand

Answer 3

RNA is complementary to the template strand, and the same as the non-template/coding strand

Question 4

RNA is synthesized in what direction

Answer 4

5’-3’, the DNA is oriented in the 3’-5’ direction

Question 5

Explain upstream vs downstream

Answer 5

first DNA base to be bound and transcried is +1, upstream takes the (-) sign and is 5’ before the +1, downstream is the 3’ direction and takes (+)

Question 6

What does the RNA polymerase holoenzyme do?

Answer 6

It binds to the DNA sequence to unwind the double helix and begin the synthesis of the RNA molecule

Question 7

Describe the 5 subunits that make up the RNA pol holoenzyme

Answer 7

Sigma binds to the consensus sequence (-35 to -10) and aids in separating the DNA so the core enzyme can tightly bind
The two alpha units assemble the enzyme
Beta is active in catalysis
Beta prime binds to the DNA
w has roles in enzyme assembly and regulation of gene expression

Question 8

Describe how RNA elongation happens

Answer 8

In the transcription bubble, RNA pol monitors the binding of free ribonucleoside triphosphate to the next exposed base of DNA - gets energy from breaking a phosphate bond
- adds ribonucleotides
last 8-9 nucleotides form a DNA-RNA hybrid

Question 9

Factor independent termination of RNA transcription

Answer 9

G-C rich region followed by a stretch of A’s which will make U in the RNA (weak bond)
G-C forms a stem loop - strong bonds
Stalling of RNA pol, reverses and runs into stem loop
knocks the RNA pol off of the DNA and terminates transcription

Question 10

Rho-dependent termination

Answer 10

Rho factor recognizes nucleotide sequences in the RNA that signal termination - it binds and slides towards the open transcription bubble and when it catches up to the polymerase (which has paused) it terminates transcription

Question 11

Steps of mRNA decay in bacteria

Answer 11

1. endonuclease cuts RNA into two pieces
2. exonuclease activity digests the RNA pieces into single nucleotides starting at their 3’ end

Question 12

What do each of the three RNA polymerases in eukaryotes transcribe?

Answer 12

RNA pol I - rRNA
RNA pol II - mRNA and some ncRNAs
RNA pol III - a variety of ncRNAs

Question 13

What is the tail of RNA pol II and what does it do?

Answer 13

Carboxy-terminal domain organizes RNA processing events - its phosphorylations serve as binding sites for many of the proteins required for:
- 5’ capping
-Splicing
-3’ cleavage and polyadenylation

Question 14

First event in eukaryotic transcription for RNA pol II

Answer 14

Binding of the TATA binding protein (TBP) to TATA sequence
- this then attracts other GTFs, including RNA pol II to the promoter
- one of the factors will open the helix into a transcription bubble

Question 15

How does the CTD in RNA pol II permit elongation?

Answer 15

When it is phosphorylated by one of the GTFs, which weakens RNA pol II bond to the preinitiation complex and allows it to move forward and transcribe RNA
- phosphorylation of the 5S serine by TFIIH serves as a binding signal for ezymes to make the 5’ cap as well

Question 16

Polyadenylation

Answer 16

CTD recognizes polyadenylation signal from the RNA and cuts off the end of the RNA (Cleavage), then adds a poly A tail (polyadenylation)

Question 17

Transcription termination for RNA pol I and III

Answer 17

RNA pol I - stopped by protein factors called terminator elements and is released
RNA pol III - terminates and dissociates after synthesizing a poly U stretch

Question 18

The two models for termination of RNA pol II

Answer 18

After the recruitment of the cleavage and polyadenylation complex (CP)
Torpedo termination model: Xrn2 pushes it off by digesting one nucleotide at a time until it reaches the pol

Allosteric termination model: Changes shape of RNA so it becomes weakly bound and just fall off

Question 19

The 3 enzymes required for capping

Answer 19

RNA triphosphatase: removes the gamma phosphate from the first nucleotide in the RNA chain

Guanylyl transferase: uses GTP as a substrate and links GMP to the first nucleotide

7-methyltransferase: adds a methyl group

Question 20

Purpose of the cap

Answer 20

Protects RNA from degredation and is required for translation (serves as a binding site for factors that mediate mRNA processing and export)

Question 21

Spliceosome

Answer 21

Contains snRNPs U1 U2 U4 U5 U6
- U1 binds to 5’ splice site
- U2 binds to branch point, and causes A to bulge out (can be bound to later when the intron folds over)
-Spliceosome assembly 4,5,6

Question 22

What are the two splicing reactions?

Answer 22

1. Between the 5’ and the internal branch of the intron. Cuts the pre-mRNA and produces an intron with a loop structure called a lariat
2. the hydroxyl group that is now at the end of the exon, reacts with the phosphate at the remaining intron-exon border and the lariat is freed and degraded (joins the two exons together)

Question 23

Alternative splicing

Answer 23

expands the proteome (set of all proteins that can be produced)
exons can be joined together in different combinations to produce different mRNAs that encode protein isoforms
most common form is exon skipping

Question 24

The two mechanisms of mRNA decay

Answer 24

Both initiated by the removal of the poly A tail
1. Cap removed by the decapping enzyme - digested 5’-3’ direction
2. Followed by digestion in 3’-5’ by exonuclease called to exosomem- then hydrolysis of the cap takes place