Molecular Biology Lecture 11 PDF

Molecular Biology – Lecture 11 Biol 367 – Molecular Biology Text: Molecular Biology: Principles and Practice (2e) Instructor: Dr. David Kwan 1  Transcription in prokaryotes Basic mechanism of tran...

Molecular Biology – Lecture 11 Biol 367 – Molecular Biology Text: Molecular Biology: Principles and Practice (2e) Instructor: Dr. David Kwan 1  Transcription in prokaryotes Basic mechanism of transcription in prokaryotes 2  Outline Bacterial RNA polymerase subunit organization – Section 15.1 Promoter sequences and promoter binding – Section 15.2 Stages of transcription (initiation, elongation, and termination) – Section 15.2 3  Transcription overview Transcription is the DNA-templated synthesis of RNA The immediate product of transcription from a DNA template is the primary transcript: – Pre-mRNA (in eukaryotes) is processed to become mRNA Figure 15-1 – Primary transcripts may be cleaved into tRNA or rRNA premrna primarytranscriptsfurther Figure 15-5 processedintosmallerfragment 4  The enzyme mechanism of RNA synthesis of tRNA rRNA Ribonucleotide is added from OH OH an rNTP to growing transcript OH OH through formation of a phosphodiester bond Direction of synthesis is 5′→3′ Catalyzed by a polymerase enzyme: RNA polymerase RNApolymerase catalyzes process Figure 15-3 5  E. coli RNA polymerase RNA polymerase enzyme isolated from E. coli consists of several polypeptide subunits The  subunit can be separated from the “core enzyme” (consisting of a subunit, ′ subunit, two  subunits, and an subunit) Together, core enzyme and  subunit form a “holoenzyme” Lisigmafactorassociate w – There are different  subunits that can associate with the coreenzyme core enzyme to form a holoenzyme (70 is the most common in E. coli) holoenzyme Figure 15-4 6 Subunits of E. coli RNA polymerase ’ – 160000 Da  – 150000 Da  – 2 x 40000 Da  – 10000 Da  – 70000 Da 7  Sigma factors and promoters Presence of the -subunit allows recognition of authentic RNA polymerase binding sites o recognition ofpromoters RNA polymerase binding sites are called promoters Transcription that begins at promoters is specific, directed by the -subunit 8  Filter binding assay (nitrocellulose) Experiment: Step 1 – mix radioactive T7 DNA and RNA polymerase, wait a few minutes Step 2 – add excess non radioactive T7 DNA Step 3 – follow polymerase DNA complexes over time using the filter binding assay shown below Filterbindingassay when nitrocellulose filter Naked DNA passthrough Assay protein-DNA 4protein binding by measuring the amount of label interacts retained on the filter w filter balled RNApolymerase entries hagetdfkh.in 9  11 4 Binding of RNA Polymerase to Promoters m How tightly does core enzyme versus holoenzyme bind DNA? 4remain tightly Experiment measures binding of DNA to boundevenw axial enzyme using nitrocellulose filters DNAduring time Radioactively labeled promoter DNA cometunboundfrom incubated with enzyme then chased with non- a coreenzymeveryquickly when added radioactive promoter DNA unlabeled Yes – Tight binding prevents displacement of radioactive DNA factorallows holoenzyme tobindtightlytopromoter recognize promotersite stays tightlybound 10  Temperature and promoter binding of E. coli RNA polymerase holoenzyme w Holoenzyme As temperature is lowered, the binding of RNA polymerase (holoenzyme) to DNA decreases dramatically Higher temperature promotes tighter DNA binding. Reason, DNA melting promotes strong binding 11  Polymerase/Promoter Binding transiently bind unbind Holoenzyme transiently binds DNA till holoenzymefinds again and again until it finds a genuinepromoter promoter – “scans” along the DNA Holoenzyme binds promoter DNA loosely at first. Complex loosely closed looselybound bound at promoter = closed promoter complex, dsDNA in closed form processbinding protein conformational pchange Holoenzyme melts ofprotein DNAcomplex DNA at promoter forming open promoter complex— polymerase tightly bound open tightly bound 1 – -factor involved in tight binding and transcription initiation Holoenzyme unwindsDNA 12  Promoter sequences Promoters are sequences of DNA recognized by RNA polymerase holoenzyme Core promoter elements are recognized by the  subunit o -10 region is 10 bp upstream of transcription start site Mediated o -35 region is 35 bp upstream of Osubun transcription start site by Figure 15-10 o UP elements found on some promoters of highly expressed genes are bound by α subunit 13  Core promoter elements Core promoter elements at -10 and -35 regions are necessary for recognition by the holoenzyme Bound by one of the various  factors that can associate with the core enzyme to form a holoenzyme ( is most common in E. coli) Promoters recognized by  not Figure 15-10 identical but have consensus sequence – TATAAT for the -10 region (Pribnow box) What is a consensus sequence? – TTGACA for -35 region sequence getfor commonnucleotide at at pos I commonnucleotide 10region t 14  I 91 pos2 A pos 3 7 in Miura, C. et al. (2015) Sci. Rep. 5:11893 15  Promoter Strength Consensus sequences (in E. coli): – -10 box sequence approximates TAtAaT – -35 box sequence approximates TTGACa Mutations that weaken promoter binding by RNA polymerase: – Down mutations differentthanconsensussequence – Usually increase deviation from the consensus sequence Mutations that strengthen promoter binding: – Up mutations closely consensus to sequence – Usually decrease deviation from the consensus sequence Direct analysis of protein-DNA interactions can identify where (what sequence) sigma factors bind – Can use DNase footprinting as a method to analyze protein-DNA Figure 15-11 interactions 16  Enlistment DNase footprinting ttstandatbnaaedeterminenowtar I ppp j.fi awayprotein bindstoDNA 10135regions Temptrein DNAinteraction prevent protectfromcleavage Sites at which protein binds DNA are protected from digestion by DNAse Labeling one end of a piece of DNA, bind protein and treat with DNAse – Can infer the position where protein binds by measuring the length of labeled fragments produced Highlight 20-1 (Fig.2a, p. 701) 17  Different sigma factors specify binding of holoenzyme to different promoters Figure 15-12 Different sigma factors recognize different consensus sequences in the core promoter elements – Determined experimentally by protein-DNA interaction studies (DNase footprinting) Different sigma factors may dominate in the cell under different conditions—by using different σ subunits, the cell can coordinate expression of sets of genes, permitting major changes in cell physiology Sources: For 70 family (RpoD, RpoS, RpoF, RpoH) and 54 (RpoN) consensus sequences: M. M. Wöstena, FEMS Microbiol. Rev. 22:127-150, online January 17, 2006. 18 For 24 (RpoE): K.M. Thompson, V.A., Rhodius, and S Gottesman, J. Bacteriol. 189:4243-4256, 2007 online, April 6, 2007, doi: 10.1128/JB.00020-07  Upstream Promoter (UP) elements In a few cases, for gene expression needed at high levels, the consensus sequence for the promoter -10 and -35 elements is not strong enough Upstream promoter element (UP element) is a promoter element Figure 15-10 that can stimulate transcription by a factor of 30 19  Modeling the Function of the C-Terminal Domain of the -subunit RNA polymerase binds to a core promoter via its -factor Binds to a promoter with an UP element through interactions with the -subunit Results in very strong interaction between polymerase and promoter This produces a high level of Cterminal domainsof α subunit transcription bindsto upelement 20 Footprinting an E. coli promoter including core elements and an UP element DNA footprinting of the template strand using alpha subunits (left) and of the nontemplate strand using alpha subunits and RNA polymerase holoenzyme (right) 21  Stages of transcription Transcription 1 initiation III. Transcription termination 2 elongation 3termination Figure 15-7 I. Transcription initiation II. Transcription elongation 22  The Role of sigma () in transcription initiation  -factor involved in specificity of gene transcription by causing tight binding between RNA polymerase and promoters Tight binding depends on local melting of DNA that permits open promoter complex  dissociates from core after enabling polymerase-promoter binding 23  Stages of Transcription: Initiation Formation of a closed promoter complex RNA polymerase holoenzyme DNA melts, converting the closed promoter complex to an open promoter complex Polymerize early nucleotides – polymerase stuck at the promoter (abortive transcription) starts stalls Promoter clearance – transcript becomes long enough to form a stable hybrid with template 4 ofactordissociates oncetranscriptioninitiationiscompleteFigure 15-13a leaving coreenzyme to transcriberest 24  Promoter Clearance downstream DNAbeing drawninto promoter wo promotermoving afterstoring enoughenergyprovid DNA scrunching mechanism. During initial transcription, RNA polymerase (RNAP) remains stationary on the promoter and bydNtp releasing unwinds and reels in downstream DNA RNApolymerase Continuetranscriptio The E.coli polymerase carries out “abortive transcription” during initiation, which causes “scrunching”: drawing downstream DNA into the polymerase without actually moving or losing its grip on promoter DNA The scrunched DNA could store enough energy to allow the polymerase to break its bonds to the promoter and begin productive transcription 25  Reuse of -factor ( cycle)  can be recycled for additional use in a process called the  cycle  can be released from the holoenzyme and released  can associate with a core enzyme In addition to primary -factor, bacteria have alternative -factors that allow expression of specialized genes by directing holoenzyme to distinct promoters 26  Stages of transcription III. Transcription termination Figure 15-7 I. Transcription initiation II. Transcription elongation 27  Stages of Transcription: Elongation After transcription initiation is accomplished and RNA polymerase “escapes” promoter, core enzyme continues to elongate the RNA ( dissociates, reused for initiation) Nucleotides are added sequentially, one after another in the process of elongation – 5’→3’ direction, RNA polymerase is processive (not released until transcription is Figure 15-14 finished) 28  Function of the Core Polymerase Core polymerase contains the RNA synthesizing machinery Phosphodiester bond formation involves the - and ’-subunits These subunits also participate in DNA binding d Assembly of the core polymerase is a major role of the -subunit 29  Transcription bubble and RNA-DNA Hybrid ~17 bp of DNA duplex is unwound, forming a “transcription bubble” enabling RNA polymerase to access the template strand The length of RNA-DNA hybridization within the E. coli elongation complex Topoisomerases act here extends about 8 or 9 bases from the 3’ end of the nascent RNA The unwinding as the polymerase moves causes positive supercoils ahead of the polymerase and negative supercoils behind the polymerase – The positive and negative supercoils are removed by topoisomerases Figures 15-6 30  Topology of Elongation Elongation of transcription involves polymerization of nucleotides as the RNA polymerase travels along the template DNA Polymerase maintains a short, melted region of template DNA DNA must unwind ahead of the advancing polymerase and close up behind it Strain introduced into the template DNA is relaxed by topoisomerases 31  Stages of transcription III. Transcription termination Figure 15-7 I. Transcription initiation II. Transcription elongation 32  Termination of Transcription There are 2 main types of terminators Intrinsic terminators function with the RNA polymerase by itself without help from other proteins Other type depends on auxiliary factor called rho (), these are therefore called -dependent terminators 1 Intrinsicterminators RNA polymerase 2 p dependent terminators auxiliaryfactor rho p 33  Rho-Independent Termination Intrinsic or rho-independent termination depends on terminators of 2 elements: – Inverted repeat followed immediately by – T-rich region in nontemplate strand of the gene An inverted repeat predisposes a transcript to form a hairpin structure A string of incorporated U causes RNA polymerase to pause stringofUs causesRNA poltopanic – If a string of U’s are incorporated just downstream of hairpin, transcription is terminated Figure 15-17a 34  Inverted Repeats (dsDNA) and Hairpins (transcripts that form RNA secondary structure) Estefomplementary bpwitself DNA forms an inverted repeat (in blue) hairpinstructure A transcript of this sequence is self- destabilize pulls complementary itself RNAawayfrom bpw – Bases can pair up to form a hairpin as seen in the DNAtranscriptionbubb lower panel dissociated 35  Rho-dependent termination: Mechanism of Rho When transcript lengthens to include rho-utilization Lovingdowncatchingupto RNA site (rut) site, rho (ρ) binds transcript polymoral Rho actively feeds the RNA through itself (driven by ATP-hydrolysis) tothatin RNA PO Rho migrates 5′→3′ along mRNA to polymerase then ah causes separation of mRNA from polymerase Iseparatio Figure 15-17b 36  Homework Problem set will be posted for the following week’s tutorial Readings – Read Ch. 15 pages 519 - 536 Extra problems to study – Solve Ch. 15 problems 2, 3, 5, 12 37