RNA Structure –Exon/intron –Cap & tail –Secondary structure Synthesis –Promoter complex –Transcription complex –Splicing Regulation –Promoter elements –Enhancer elements –Chromatin structure

RNA structure rRNA-ribosomal tRNA-transfer snRNA-small nuclear miRNA-”micro” regulatory mRNA-messenger –Translated region –5’ and 3’ untranslated region –Poly-A tail –7mG cap

RNA structure Nucleic acid Base pairing Accessory proteins 2 0 structure of HCV RNA 2 0 structure of tRNA

Gene structure Promoter/Initiator –Genes are read 5’ to 3’ –TATA box TATAAAA – Pyrimidine-rich Initator (INR) c/t c/t A n a/t c/t c/t –Gene specific promoter Intron –Enhancer/repressor –Arrest points Exon Intron CoreEnhancer Promoter Position Structure

mRNA synthesis and processing Initiation –Promoter –Enhancer/repressor Transcription –Elongation –Pause/arrest Termination Processing –5’ cap, 3’ poly-adenylation –Splicing

RNA Transcription Pre-Initiation Complex (PIC) binds promoter PIC recruits RNA Polymerase II PolII transcription elongation complex (TEC) transcribes sequence 7’-methyl-guanosine cap Spliceosome ribozymes remove introns Polyadenylation factors recruit poly-A polymerase

Pre-initiation complex Transcription Factor IID (TFIID) –TBP TATA box –11 other subunits TFIIA –Stability –Blocks repressor proteins TFIIB –Defines transcription start –Recruits TFIIF/Pol II TFIID TFIIB TATA Inr TFIIA

Pre-Initiation Complex TFIIE & TFIIH –Helicase –cdk activity –Carboxy-Terminal Domain (CTD) kinase Phosphorylates S5 Pol II Start trigger

Pol II Binds DNA above cleft, melts, and settles on active site –Transcription bubble of ~10bp Growing RNA exits via saddle Melted DNA strands exit through clamp Coding Complimentary RNA Upstream DNA Entry RNA Exit Clamp

Active site Non-specific recruitment “test fit” via PO 4 Template match aligns PolII for catalysis

Transition to Elongation Early instability –Abortive cycling –Upstream slip –Escape commitment Mobility of short RNA fragments Phosphorylation of S2 in CTD Dissociation of TFII D, A, B

Abortive initiation Full-length product 3 different bacterial polymerases 3nt fragment 9nt fragment 13nt fragment

Critical events 1.Separation from DNA template: 9nt 2.Threading the saddle: 15nt 3.23 nt complex –Annealing of elongation factors –5’ capping 1 2 3

Elongation Proper Pol II is sufficient 200 nt/s FACT, TFIIF cofactors, 1000 nt/s TFIIS proofreading

Termination Pol II transcribes past poly-A signal (AAUAAA) Recruitment of polyadenylation factors –Poly-A polymerase remains associated w/TEC –Cleavage and polyadenylation specificity factor (CPSF) binds AAUAAA Dissociation from DNA requires –PAD-associated P-TEF dissociation –Pol II CTD S2 dephosphorylation –Dissociation of PAF & TFIIS

RNA Processing Raw transcript “heterogeneous nuclear RNA” 7’methyl guanine Cap –Capping enzymes part of PIC –5’ base is backwards –Stability –Translational efficiency 7’ methylated guanine (methylated after linkage) 5’-5’ linkage

Nuclear Structure & Transcription Nucleolus Cajal bodies Transcriptosome/B-Snurposome/Speckle Handwerger & Gall 2006

Splicing Sequence specific –5’-AG|GUAAGU… –A(Py) 20 CAG|G-3’ Loop/lariat formation –2’ trans-esterification forms lariat Only RNA nucleotides have 2’ hydroxyls –3’ transesterification excises intron Spliceosome is part of TEC

Lariat formation mRNA repair Spliced mRNA Free lariat 2’ Carbon 3’ Carbon Hang et al 2015

Yan et al 2015 Frankenstein et al 2012 Spliceosome U1 associates with TEC –Recognizes 5’ splice sequence U2 is recruited to nascent intron –Recognizes branch point U4/U5/U6 is recruited –U4 chaperone –U6 forms loop –U5 binds exon ends

Spliceosome Specificity from spliceosome RNA base pairing Splice definition prior to termination, but not always excised immediately Alternative splicing

Nuclear Export Analogous to protein export (but no ranGTP) Nuclear eXport Factor targets mRNP –Exon-exon junction complex –Only processed RNA Transport through Nuclear Pore Complex (Protein export model)

Degradation 3’ de-adenylation –Crc4p/Pop2p –Exosome mediated degradation snRNA/snoRNA/rRNA processing Decapping Degradation –Missense –Targeted

Poly(A) Binding Protein Binds A Shield mRNA from degradation –Decapping after poly(A) drops to 10 –Inhibition of deadenylation –Stabilization of 5’cap (eIF4F) Promote translation –Recruits 40S –Reinitiation