OBJ - RNA Synthesis and Processing

Question 1

Define transcription.

Answer 1

Transcription is the first step in gene expression where a particular segment of DNA is copied into RNA by RNA Polymerase

Question 2

Explain in molecular detail the primary catalytic activity of RNA polymerase.

Answer 2

RNA Polymerase is an enzyme that produces the primary transcript RNA - 1st draft

Needs promoters to tell it where to start & stop codons to tell it to terminate

Produces:

• mRNA - transports message to rRNA
• tRNA - template for building proteins
• rRNA - makes up ribosomes

Pre-initiation:
requires a core promoter/Transcription Start Site (TSS) - “TATA box”

Initiation
PRO: binding of RNAP to promoter as coenzyme with 5 subunits
EUK: RNAP doesn’t directly bind, needs transcription factors to mediate binding of RNA Polymerase & unwinds double helix

Elongation:
RNA Polymerase moves along template strant from 3’->5’ (net making 5’-3’ of daughter strand)

Termination:
EUK: Polyadenylation/Poly A Tail
PRO:
- Rho INdependent = hairpin loop & a run of U’s that weakens bonds & pulls Poly U trasncript out of active site, thus terminating Trasncription
- Rho dependent termination = Rho destabilizes interaction between mRNA & template; releasing/pulling it out

Question 3

List the specificities of the eukaryotic RNA polymerases.

Answer 3

PRO: 1 type of RNAP makes all 3 types of RNA

EUK:
*no proofreading function
-initiates chains
RNAP I => synth pre-rRNA (most prevalent)
RNAP II => synth mRNA (opens DNA at 
                   promoter site)
RNAP III=> synth tRNA & small rRNAs

Question 4

Explain how pre-mRNA is modified in eukaryotes after transcription, including an explanation of the sequence features required for pre-mRNA splicing and the mechanism of the process.

Answer 4

Immature single strand of mRNA that has all exons & introns

Exons - coding segment that remain part of protein
Introns - coding sequences that are removed by spliceosome

Add 5’ Cap of 7-Methylguanosine
Adds Poly-A Tail to 3’; ~200 A’s
Splicing out of introns
=mRNA

Comprises the bulk to hnRNA (heterogeneous nuclear RNA)

1) hnRNA = initial transcript that is to become mRNA combines with snRNPs (small nuclear ribonucleoproteins) to create SPLICEOSOME
2) Lariat-shaped (looped) intermediate is formed
3) Lariat is released & 2 exons joined by transesterification

Question 5

Explain how tRNAs are processed following transcription.

Answer 5

Structure:

• cloverleaf form with an anticodon loops
• 3’ end has CCA (3’-5’) that is the AA acceptor site

Charging
- uses ATP to transfer AA to tRNA & form peptide bond

tRNA Wobble

• only 1st 2 base pairs necessary, the 3rd is a “wobble” position due to genetic code degeneracy
• 3rd position – not TERRIBLY important – mostly interchangeable;
• Inosine (A,C,U) & G = U