RNA Metabolism Transcription and Processing CH353 April 1, 2008

Types of RNA Present in all cells, mitochondria and chloroplasts Messenger RNA (mRNA) – encode protein sequences from DNA Transfer RNA (tRNA) – decode mRNA sequences; activate specific amino acids for protein synthesis Ribosomal RNA (rRNA) – catalytic components of ribosomes; for tRNA binding, codon-anticodon recognition, peptidyl transfer Present predominantly in eukaryotic cells Catalytic RNAs – components of ribonucleoprotein enzymes 7S RNA – component of signal recognition particle for secretion Small nuclear RNAs (snRNAs) – spliceosome subunit components Small nucleolar RNAs (snoRNAs) – guides for rRNA modification MicroRNAs (miRNAs) – silencing gene expression

RNA Polymerase Reaction General reaction: (NMP)n + NTP → (NMP)n+1 + PPi Template + n NTP → Template + ppp(NMP)n + n PPi Requires DNA template, NTP’s and Mg2+ (Initiator NTP is primer) Reaction driven by hydrolysis of PPi; ∆G’º = -19 kJ/mol Mechanism: nucleophilic attack by 3’-OH on α-phosphate of NTP with PPi as leaving group

Basic Properties of Transcription Transcription from DNA without strand separation Transient transcription bubble of single stranded DNA Transcription synthesizes single stranded RNA Transient RNA-DNA hybrid intermediate Transcription is specific to DNA strand Template strand DNA transcribed; reverse complement of RNA Nontemplate or coding strand of DNA; same sense as RNA Transcription has an initiation site on DNA RNA synthesis begins at promoters no primer required: GTP + NTP → (5’) pppGpN-OH (3’) + PPi Transcription has a termination site RNA synthesis ends at terminators; defines DNA template

Template and Nontemplate Strands Transcription Units of Adenovirus Genome Nontemplate sequences same sense as RNA transcripts Templates for RNA synthesis do not include entire DNA strand could be on either or both DNA strands A sequence and its complement do not both encode proteins

Transcription with E. coli RNA Polymerase Topoisomerase I Topoisomerase II Transcription bubble ~17 bp Rewinding Unwinding Nontemplate strand Template strand RNA-DNA hybrid ~8 bp

Properties of E. coli RNA Polymerase Large complex (Mr 390,000) of 5 core subunits (a2bb’w) RNA polymerase holoenzyme has additional s subunit required for specific initiation Most common is s70 (Mr 70,000) for recognition of most promoters; s32 for heat shock promoters; s54 for regulation by enhancers (NtrC) RNA polymerases lack 3’ → 5’ exonuclease proofreading activity; can remove incorrect nucleotide by reversal of polymerase reaction Error rate: 10-4 to 10-5 RNA polymerases are highly processive; dissociation of polymerase from DNA terminates transcription RNA polymerization rate: 50 – 90 nucleotides / second (comparable to DNA polymerase II); transcription of most genes in < 1 minute

Promoters for E. coli RNA Polymerase Recognized by holoenzyme with s70 subunit (most common) Alignment of RNA start sites reveals upstream consensus sequences TTGACA at -35 and TATAAT at -10 relative to start (+1) Strong promoters have consensus sequences and additional A-T rich UP element (between -40 and -60)

Transcription with E. coli RNA Polymerase Initiation and Elongation E. coli RNA polymerase holoenzyme binds to promoter sequence Closed complex: RNA polymerase bound but DNA still double stranded Open complex: 12-15 bp region of DNA from within -10 to +2 or +3 is unwound Transcription initiation: formation of full transcription bubble; conformational change to elongation form Elongation form: complex moves away from promoter (promoter clearance); s subunit dissociates after first 8-9 nucleotides are polymerized

Transcription with E. coli RNA Polymerase Termination Rho (r) independent (shown) transcription of sequence that can form hairpin loop; followed by AAA in template for UUU in transcript pausing of RNA polymerase allows hairpin loop formation and disruption of RNA-DNA Rho (r) dependent has CA-rich sequences in template and r binding sites on transcript the r protein has helicase activity; uses ATP for translocation on RNA

Regulation of E. coli Transcription Specific s subunits determine promoter selection for generalized changes, e.g. development stage (sporulation) or stress response (heat shock) Activation of transcription (Positive Regulation) cAMP receptor protein (CRP) involved in catabolite activation; increases transcription of gene involved in utilization of carbon sources other than glucose CRP bound to cAMP binds to DNA upstream of weak promoters Repression of transcription (Negative Regulation) lac and trp repressors block transcription by binding to operator sequences within or downstream of promoters lac repressor bound to inducer doesn’t bind to operator

Regulation of Transcription in E. coli

DNA Footprinting Analysis Used for qualitative analysis of DNA-protein binding and localizing binding sequences Uses end-labeled DNA and partial DNase digestion + bound protein Partially digested DNA is analyzed by denaturing PAGE Sequences binding to protein are protected from DNase digestion – indicated by missing bands on gel

Electrophoretic Mobility Shift Assay (EMSA) Used for quantitative analysis of DNA binding proteins DNA fragment containing a promoter region is labeled DNA probe incubated with protein fractions suspected of having binding activity Assayed fractions are analyzed by non-denaturing PAGE Electrophoretic mobility of DNA is slower if bound to a protein Arrow indicates migration of bound DNA probe EMSA of protein purification fractions Fractions containing TFIIC2 bind labeled VA1 DNA probe, slowing its mobility on non-denaturing PAGE Yoshinaga et al. (1989) J.Biol.Chem 264, 10726

Eukaryotic RNA Polymerases RNA polymerase I II III Subunits 14 12 16 unique abb’w-like 5 5* 5 common 4 4 4 unique 5 3 7 Inhibition [a-amanitin] (resistant) low high Products pre-rRNA mRNAs, tRNAs, (28S, 5.8S, 18S) 5 snRNAs U6 snRNA, 5S rRNA, 7S RNA * large subunit has carboxy terminal domain (CTD)

Properties of RNA Polymerase II RBP1, largest subunit (Mr 220,000) is homologous to E. coli RNA polymerase b’ subunit has unusual carboxyl-terminal domain (CTD) with heptad (-YSPTSPS-) repeats of 27x (yeast) or 52x (human) plus unstructured linker; CTD extends from main polymerase structure ~ 90 to 160 nm RNA polymerase II promoters Many have TATA box -35 to -26 bp from start (T-A-T-A-A/T-A-A/T-A/G) Some have initiator element instead of TATA box (Y-Y-A+1-N-T/A-Y-Y-Y); these also have a 20-50 bp CG-rich region ~100 bp upstream of start Many have promoter-proximal elements (control regions) within 100-200 upstream of start

Transcription with RNA Polymerase II Assembly of RNA polymerase and transcription factors at promoter Formation of closed complex TFIIH (12 subunits) has helicase, kinase and DNA repair activities Initiation and promoter clearance TFIIH phosphorylates CTD of Pol II causing conformation change initiating transcription; TFIIE, TFIIH released after 1st 60-70 nt RNA Elongation Elongation factors suppress pausing and coordinate RNA processing; pTEFb phosphorylates CTD Termination and Release Pol II dephosphorylated after release

Synthesis of Eukaryotic mRNA

Structure and Synthesis of mRNA Caps Most eukaryotic mRNAs have 5’ cap structure 7-methylguanosine with (+) charge linked to 5’-terminal nucleotide of mRNA by 5’,5’ triphosphate Unique structure important for translation initiation Cap formed by transfer of guanylate to 5’ diphosphate and methylation using S-adenosylmethionine

Splicing of mRNA Transcripts splice donor branch point splice acceptor Formation of Spliceosome: (5 snRNAs + 50 proteins) U1 snRNP binds at splice donor site U2 snRNP binds at branch point site (+ ATP) U4:U6 snRNP and U5 snRNP bind forming inactive splicesome (+ ATP) U4 snRNP and U1 snRNP released, U6 snRNP binds splice donor activing spliceosome (+ ATP) Splicing occurs by 2 step mechanism with lariat intron and spliced exons as products

Splicing Reaction Mechanism Mechanism for spliceosomal introns and self-splicing group II introns 2 step splicing mechanism: 2’ OH of adenosine at branch point is nucleophile for attack on splice donor phosphodiester bond; 3’ OH of 5’ exon is leaving group 3’ OH of 5’ exon (splice donor) is nucleophile for attack on splice acceptor phosphodiester bond; 3’ OH of intron is leaving group Products: spliced RNA + intron RNA with 2’,5’ branch (lariat)

Coupling Transcription and RNA Processing C-terminal domain (CTD) provides attachment sites for complexes involved with RNA processing Cap-synthesizing complex binds to CTD and 5’ end of mRNA precursor 5’ end of RNA is capped Cap-binding complex binds CTD and 5’ cap of mRNA Spliceosome components bind CTD, capturing splice donors and branch points of nascent mRNA

Termination and Polyadenylation Eukaryotic mRNA have a 3’ terminal poly(A) tail (80 – 250 nucleotides) Polyadenylation linked to transcription termination Enzyme complex binds CTD cleaving RNA at poly(A) site; between AAUAAA and GU-rich sequences Polyadenylate polymerase of enzyme complex adds poly(A) to 3’ OH: RNA + nATP → RNA-(pA)n + nPPi

Alternative Splicing of mRNA Transcripts Alternative Polyadenylation Sites Poly(A) site choice forms diversity of 3’ ends for mRNA; C-terminal ends for proteins Alternative Splicing Patterns A splice donor may have multiple splice acceptors, forming diverse mRNAs and encoded proteins

Splicing of Calcitonin/CGRP mRNA Precursor mRNA has 2 poly(A) sites: one recognized in thyroid and other recognized in brain Shorter thyroid transcript is processed at exons 1-2-3-4 encoding calcitonin – a calcium regulating hormone Longer brain transcript is processed at exons 1-2-3-5-6 encoding CGRP (calcitonin gene-related peptide) – a neurotransmitter 1 gene provides 2 different proteins depending on alternate processing of RNA

Processing of Human rRNA Precursors In the nucleolus: RNA pol I transcribes 45S pre-rRNA from multiple rRNA genes Small nucleolar RNAs (snoRNA) guide methylation and cleavage of the precursor into mature 18S, 5.8S and 28S rRNAs >100 of 14000 nucleotides are methylated

Processing of Bacterial rRNA Precursors E. coli RNA polymerase transcribes RNA from one of 7 rRNA genes Each precursor contains 16S, 23S and 5S rRNAs plus 1 – 2 tRNAs Site-specific methylation of pre-rRNA using guide RNAs Nuclease cleavage with: RNase III RNase P RNase E Processing with various specific nucleases

Processing tRNA Precursors Yeast tRNA is processed from precursor by removing the 5’ end with RNase P (a ribozyme), then the 3’ end with RNase D The terminal CCA(3’) is added 1 nucleotide at a time by the enzyme tRNA nucleotidyl transferase – template independent RNA synthesis Bases are modified during the processing of tRNA Some tRNAs are spliced, removing the intron in the last step