lecture 18- RNA Processing

Question 1

most RNA molecules are synthesized as ____

Answer 1

biologically inactive precursors (primary transcripts)

Question 2

all tRNA’s are synthesized as ___
all large rRNA’s are synthesized as ___
most of the small noncoding RNA’s are ___
all mRNA’s in eukaryotes must be ___
most of the bacterial mRNA’s are ___

Answer 2

larger pre-tRNA’s
single very large precursor
modified
modified
NOT modified

Question 3

name types of RNA processing

Answer 3

1- cutting (cleavage), trimming, splicing
2- modification of nucleotides (5’ capping & polyadenylation)
3- editing (base modification, insertion, deletion)

Question 4

rRNA processing in prokaryotes

Answer 4

bacterial ribosome (70S composed of 50S and 30S) - 30S composed of 16S RNA and proteins
- pre-rRNA transcript (30S) —> methylation —> cleavage: pre-rRNA containing all rRNA sequences and tRNA is cleaved/trimmed by endoribonucleases —> mature rRNA’s

Question 5

tRNA processing in prokaryotes

Answer 5

• the precursors are cut and trimmed by appropriate ribonucleases
• many bases in tRNA’s are also modified

Question 6

mRNA processing in prokaryotes

Answer 6

most bacterial mRNA’s are not processed b/c mRNA is already being translated by ribosome

1- monocistronic- contains info for producing one protein (5’s untranslated region, Shine Dalgarno sequence ; protein-coding region ; 3’ untranslated region, stop codon)
2- polycistronic- contains info for making more than 1 protein

Question 7

all eukaryotic transcripts are synthesized as ___ and must be ___

Answer 7

nonfunctional precursors (primary transcripts)
must be modified

Question 8

describe RNA processing for RNA polymerases in eukaryotes

Answer 8

RNA pol I and RNA pol III products are cut and trimmed, and usually edited

RNA poly II mRNA products are always modified!
- G-cap is added at 5’ end during transcription
- introns are removed during and after transcription
- polyA tail is usually added at 3’ end after transcription
- some mRNA’s are edited

Question 9

rRNA processing in eukaryotes

Answer 9

RNA poly I product synthesizes RNA in nucleolar region, 45S RNA (pre-rRNA), is processed to 5.8S, 18S, and 28S in nucleolus with an assist of snoRNA’s (small nucleolar RNA’s)
- during this processing, many bases are modified by methylation, transcript then cleaved and trimmed and spliced to 5.8S, etc.

Question 10

tRNA processing in eukaryotes

Answer 10

• both the 5’ and 3’ ends are trimmed in all tRNA’s by RNAse P and D
• CCA added to 3’ end
• introns (when present) are removed by splicing- specialized endonuclease/ligase
• some bases are modified in all tRNA’s to provide stability and enhance the functioning of the mature tRNA
  
  • uncommon bases are produced by modified tRNA bases, this always occurs at a specific site, not random

Question 11

mRNA processing in eukaryotes

Answer 11

MUST BE MODIFIED
- capping- 5’ end of nascent transcripts
- splicing- removal of introns
- cleavage- 3’ ends are generated by cleavage (NOT termination)
- some transcripts may be edited

This is all coordinated by CTD (carboxyl terminal domain) of RNA Poly II

Question 12

CTD of RNA poly II and transcript modification

Answer 12

pattern of phosphorylation dictates which molecule is bound to RNA polymerase, like:
- capping enzyme, components of splicing machinery, polyadenylation and cleavage factors

Question 13

eukaryotic mRNA’s are capped at 5’ end, b/c…

Answer 13

• protects 5’ end from exonucleases
• necessary for translation b/c cap is recognized by ribosomes
• 5’ cap: residue of 7 methylguanosine (7-meG)
• 7-meG is attached to 5’ terminal residue of every RNA via 5’-5’ triphosphate linkage
• caps may differ by methylation pattern

Question 14

guanylyltransferase is associated with CTD of RNA poly II

Answer 14

TFIIH phosphorylates CTD —> promoter clearance & recruitment of capping enzyme

1- the 1st modification state (phosphorylation by TFIIH) of CTD allows promoter clearance and recruitment of “capping enzyme”: guanylyltransferase

2- the 5’ cap is formed by condensation of a molecule f GTP with the triphosphate at the 5’ end of the transcript (5’-5’-triphosphate linkage)

3- the guanine is subsequently methylated at N-7 and additional methyl groups are often added at the 2’-hydroxyls of the first and second nucleotides adjacent to the cap

Question 15

5’ cap synthesis happens as soon as ___

Answer 15

transcription of mRNA starts
- guanylyltransferase (the capping enzyme) is associated with the Pol II CTD to ensure that each mRNA is capped as it is transcribed

• once the cap is complete, guanylyltransferase dissociates, and the cap-binding complex (CBC) binds

Question 16

termination of transcription RNA poly II

Answer 16

factors responsible for cleavage of the primary transcript bind to the AAUAAA sequence (polyadenylation sequence), resulting in cleavage somewhat downstream of that position

Question 17

generating the 3’ end of eukaryotic mRNA

Answer 17

CPSF= Cleavage & Polyadenylation Specificity Factor
CstF = Cleavage stimulation factor

• CPSF & CstF are recruited to PolyA signal sequence (AAUAAA) from CTD
• endonucleases cut just upstream from GU-rich sequence (mRNA cleavage)

Question 18

addition of the 3’ polyA tail to the transcript for eukaryotic mRNA

Answer 18

• 3’ polyA tail, typically 80-250 adenine residues
• serves as a binding site for one or more specific proteins that help protect mRNA from enzymatic destruction
• PAP (poly A Polymerase) adds a stretch of adenines to the 3’ generating a polyA tail postranscriptionally
• Poly A Binding Proteins (PABPs) bind to PolyA tail protecting it from 3’ to 5’ exonucleases (degradation)