RNA

Central Dogma:

DNA Þ transcription to RNA Þ translation to protein

Overview of Gene Expression

RNA polymerase makes RNA: one in prokaryotes, three in eukaryotes

RNA polymerase does not need a primer like DNA polymerase – due to presence of 2’ hydroxyl group

Rifamprin – attacks prokaryotes RNA polymerase

In Prokaryotes: transcription and translation within same cellular compartment at same time

In Eukaryotes: RNA and protein synthesis are separated in time and space

Fundamentals of Transcription

DNA:

Pre-mRNA:

mRNA:

5’ untranslated region (CAP)-AUG-coding region-stop-(polyA tail) 3’ untranslated region

Initiation: promoter recognition

How did they derive these consensus sequences?

Elongation: RNA synthesis

DNA unwound, "transcription bubble" produced, transcription occurs off coding strand which is displaced as it is elongated

Termination:

prok: 3' end formed by stopping; RNA poly reaches a stop sequence (terminator) and dissociates from the template
euk: 3' end formed by processing rather than stopping; RNA poly continues to transcribe beyond what will become the mature 3' end, terminating at undefined sites in the 3' flanking sequence

Capping: post-transcriptional modification

CAP structure: 7-methyl-guanosine-PO₄-PO₄-PO₄-5’ end of RNA (the 1^st couple 2’OH Þ 2’OCH₃)

has a 5’-5’ triphosphate bridge

required for protein synthesis

serves to stabilize mRNA

Polyadenylation:

begins with adenylation signal (AUUAAA) – cleavage endonuclease) occurs here, 100-150 adenines added by polyA polymerase
purpose of poly A? – protect from 3' endonuclease; signal for translation; mRNA stabilization; facilitates transport from nucleus to the cytoplasm

Splicing – introns are removed, connecting 5’ splice site to 3’ splice site

cis-acting signals: GU at 5’ splice site, AG at 3’ splice site, A somewhere in the middle – branch point Þ precedes the polypyrimidine tract (rich in U and C)

trans-acting factors: U1, U2, U4, U5, U6 and other proteins (50 – 100)

mechanisms of splicing: 2 steps – transesterification

(1) 2’ OH of branch point A attacks 5’ splice site, producing lariat intermediate
(2) 3’ OH of 3’ splice site (AG) ligates to 5’ splice site (GU), releasing lariat

why do you want splicing? – evolutionary plasticity (flexibility at DNA level); ability to do alternative splicing