Nucleic Acids 17 Regulatory RNAs Prokaryotes PDF

BIOC 3041 Nucleic Acids Biochemistry Regulatory RNAs Prokaryotic Examples Recap of lecture • Eukaryotic promoter contain subsets of four different sequences • Prokaryotic σ factor replaced by many eukaryotic GTFs that assist RNA Pol • Prereplicative complex involves sequential TFII assembly...

BIOC 3041 Nucleic Acids Biochemistry Regulatory RNAs Prokaryotic Examples Recap of lecture • Eukaryotic promoter contain subsets of four different sequences • Prokaryotic σ factor replaced by many eukaryotic GTFs that assist RNA Pol • Prereplicative complex involves sequential TFII assembly followed by phosphorylation events • Eukaryotic RNA Pol II targeted by multiple protein kinases for promoter escape • TATA-Binding Protein (TBP) bends DNA minor groove almost flat with β-sheet • RNA Pol II enzyme needs to be recruited, get chromatin unwound for action • Multisubunit Mediator complexes generally needed for RNA Pol activity • RNA Pol II phosphorylation serves as switch between initiation and elongation • Elongation factor proteins accelerate RNA growth by stopping pauses (errors..) • FACT-mediated reversible histone removal, replacement keeps DNA packed & protected • Proper elongation, termination, and processing requires enzyme recruitment • RNA capped early to protect transcript and ensure proper processing • RNA 5’ capping needs GTP, SAdoMet and three sequential enzymes • RNA’s poly-A tail partly encoded by DNA, RNA cleaved, enzymes add extra A • Transcriptional termination models differ in that one needs RNase (torpedo) • The RNA gene transcribing RNA Pol I, III each require specialized GTFs • www.youtube.com/watch?feature=player_detailpage&v=DoSRu15VtdM&list=PLB873E26ADA0E157B Regulatory RNAs • RNAs exploit Watson-Crick base-pairing to other RNAs, DNAs • ..block binding • • …..alter RNA structure ….recruit protein • Transcribed RNAs may be enzymatically altered to ‘mature’ RNAs • RNAs often bind proteins, even metabolites, to alter metabolism RNA’s single stranded properties facilitate regulatory roles Regulatory RNAs in Bacteria • Bacterial small RNAs can regulate gene expression • Transcription can be controlled, e.g., 6S RNA binds RNA Pol, down-regulates transcription • 6S RNA builds up in growing E. coli cells as they enter stationary phase where nutrients become depleted and cell division then stops (stress!) • Stationary phase causes alternative σS factor to be made, outcompetes σ70 subunit, and σS factor promotes transcription of survival proteins • Small RNAs (sRNAs, 80-110 nucleotides) made from tiny genes bind to complementary target mRNAs to form double-stranded RNAs • sRNAs therefore cause mRNAs destruction by RNase, blocking translation (but sRNAs can in some cases stimulate translation…) sRNAs can serve to repress levels of one protein and promote another Regulatory RNAs in Bacteria-2 • Hfq$ (Host Factor Q protein) RNA chaperone can helps sRNA bind mRNA target • Hfq aids binding of short, imperfect matching mRNA-sRNA and then stabilizes it (not very stable since not many base pairs…) • σS factor (S=Stationary phase) translation is affected by this mRNA-sRNA binding, either stimulated or inhibited RBS=Ribosome Binding Site • Translation is activated by binding & removing a RBS-blocking RNA region • sRNA binding/masking RBS site blocks ribosomes, mRNA translation • Small antisense RNAs may be transcribed, bind to and block target mRNAs Bacterial mRNA translation can be regulated by sRNA binding to key regions Riboswitches Are In Gene Transcripts Whose Expression They Control Through Changes in Secondary Structure • “Ribo-switches” are “built-in” metabolite sensors using RNA sequences • controls mRNA translation in response to changes in metabolite amount Two components: 1) Apatamer – 5′ untranslated region (UTR) region that binds a metabolite 2) Expression platform - • Apatamer binds metabolite and changes conformation, causing a change in the secondary structure of the downstream expression platform Riboswitch forms spontaneous RNA base-pairs & structures after gene transcription 70-200 nucleotides long • Results in altered transcription, translation, or splicing of RNA A gene’s transcripts can directly sense metabolite concentration changes Riboswitches Are In Gene Transcripts Whose Expression They Control Through Changes in Secondary Structure - 2 • Riboswitches usually upstream of genes involved in synthesis of metabolite that the riboswitch responds to • S-adenosylmethionine (SAM)-sensing riboswitch found in Methionine–utilizing genes • Binding of SAM causes one of two different rearrangements in aptamer expression platform shapes change too “Attenuation” Causes RNA Pol dissocation • Attenuation blocks any further RNA Polymerase transcription of the transcript • 2nd example blocks any ribosomal translation of the transcript initiation prevented by making a structure with the RBS site www.youtube.com/watch?v=xdTlfj5qFrE Activated riboswitch structures block further transcription or any translation Riboswitches Are In Gene Transcripts Whose Expression They Control Through Changes in Secondary Structure - 3 • Different outcomes of metabolite binding to riboswitches; • Conformational change in effector domain activates or prevents transcription termination • Blocks or increases access to ribosome binding site • Riboswitches exhibit protein-like structure diversity Riboswitch structures alter ribosome interactions GMP queosine SAM Riboswitches Respond to Several Metabolites • Riboswitches can bind ~19 recognized metabolites, each with unique structures • Riboswitches can also respond to uncharged tRNAs through the aminoacyl tRNA synthetase gene • When charged tRNA amount falls, uncharged tRNAs (only!) can bind to the riboswitch structure and cause it to flip from “off” to “on” (forms anti-termination structure) RBS Uncharged tRNA RBS Riboswitches can bind and thus respond to simple and complex metabolites Lecture Recap • RNA’s single stranded properties facilitate regulatory roles • RNAs can serve as regulators of gene transcription and translation • sRNAs can serve to repress levels of one protein and promote another • Bacterial mRNA translation can be regulated by sRNA binding to key regions • A gene’s transcripts can directly sense metabolite concentration changes • Activated riboswitch structures block further transcription or any translation • Riboswitch structures alter ribosome interactions • Riboswitches can bind and thus respond to simple and complex metabolites Reading – MBPGFG p 520-7, 558-60, MBOG7 pg 701-6, MBOG6 pg 633-657 • Midterm2 is next class • Midterm covers Lecture 9 DNA replication machinery to last Friday’s Eukaryotic Transcription lectures • Expect 8 T/F, ~7 M.C., ~7 short answer, and a choice of 3 of 6 longer answer questions