BIOL10221 Molecular Biology Lecture Notes PDF

BIOL10221 “Molecular Biology” Dr David Boam Module 5 - Gene expression in Eukaryotes Lecture 2: RNA Processing BIOL10221 lecture 6/2  RNA Processing – Introduction to eukaryotic mRNA processing mechanisms – Importance for regulation and diversi...

BIOL10221 “Molecular Biology” Dr David Boam Module 5 - Gene expression in Eukaryotes Lecture 2: RNA Processing BIOL10221 lecture 6/2  RNA Processing – Introduction to eukaryotic mRNA processing mechanisms – Importance for regulation and diversity By the end of the lecture you should understand main processing reactions and how they are carried out Capping Polyadenylation Splicing Splicing as a means of generating multiple functions from one gene BIOL10221 lecture 6/2  Steps in mRNA Processing Gene Transcription Cleavage/ Cappin Polyadenylatio Nucleus CAP g n AAAAAAAA Splicing CAP AAAAAAAA Mature messenger RNA CAP BIOL10221 lecture 6/2AAAAAAAA  mRNA capping GTP G CH3 G P P P B P P P 5' 5' 5' CH3 3' 3' 3' O 5' to 5' via the phosphates - not the P B normal 5’ phosphate - 3'OH link 5' 3' O CH3 Methylation at 2' position of first 2 P nucleotides and on the added G Linked to transcription initiation Increases stability of mRNA OH Required for efficient splicing Nuclear export and Translation initiation BIOL10221 lecture 6/2  3’ Cleavage and Polyadenylation AAUAA CA GU Signals for cleavage/ polyadenylation polyA polymerase AAUAAA, G/U or U rich region and cleavage factors Steps in process: 1) Cleavage and Polyadenylation CPSF CstF specificity Factor (CPSF) binds to AAUAA CA GU AAUAA 2) Cleavage stimulatory factor (CstF) binds to G/U, - recruits cleaving factors and Poly- A polymerase (PAP) AAUAA C-AAAAAAAAAAA 3) Cleavage and addition of polyA PolyA tailing functionally linked to transcription Major role in stability BIOL10221 lectureand 6/2 translation  Pre-mRNA splicing – anatomy of an intron Signals for splicing: Conserved sequences at the Y= pyrimidine 5’ splice site R= purine 3’ splice site N= any branchpoint region recognised by factors which remove introns 5’ (donor) site branch point3’ (acceptor) site 5’ AG GU RAGU YNYURAY YAG GU 3’ exon intron exon 15-30 bases BIOL10221 lecture 6/2  Splicing is a two step mechanism Step one The Two Steps of Splicin g Cleavage at 5’ splice site 1 pGU A p 2 and lariat formation at Branchpoint sequence 1 OH A p 2 Step two 1 p 2 Cleavage at 3’ splice site Removal of intron region A OH and exon ligation BIOL10221 lecture 6/2 Spliceosome assembly pathway Splicing carried out by small nuclear ribonucleoprotein particles (snRNP’s or snurps) and protein splicing factors snRNP = small nuclear RNA (snRNA) + numerous proteins snRNP’s involved in splicing are U1, U2, U4 U5 and U6 Stepwise assembly to form a “spliceosome” U2, U5 and U6 = the active site of the spliceosome BIOL10221 lecture 6/2 Spliceosome assembly pathway U1 – splice donor +U2 - branchpoint +U4/5/6 - displace U1 Rearrange – U2/5/6 “active site” BIOL10221 lecture 6/2  RNA processing - summary Main themes – 3 processing events: 5’ capping 3’ cleavage and polyadenylation Pre-mRNA splicing – Sequential recruitment of protein factors - macromolecular assemblies – Functional link to transcription – Catalytic RNA – RNA -protein and RNA-RNA interactions Function – Increase stability and translational efficiency – Splicing requires catalytic RNA – Splicing and editing increase the number of possible products arising from one gene – BIOL10221 lecture 6/2  Alternative splicing Gene mRNA splicing Exon inclusion/exclusion – proteins with altered sequence or truncated if included exon has a stop codon Examples – Sex determination in fruit flies – Control of Flowering Time – Olfactory receptor diversity Different proteins (isoforms) can be produced from a single gene Isoforms may have different functions BIOL10221 lecture 6/2  RNA processing - summary Main themes – Sequential recruitment of protein factors - macromolecular assemblies – Catalytic RNA – RNA - protein and RNA-RNA interactions Function – Increase stability and translational efficiency – Splicing requires catalytic RNA – Alternative splicing increases the number of possible products arising from one gene – BIOL10221 lecture 6/2  RNA Processing What you should understand – main processing reactions and how they are carried out Capping Polyadenylation Splicing – Splicing as a means of generating multiple functions from one gene BIOL10221 lecture 6/2