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RNA Polymerase Molecules Proceeding at a Maximal Speed of About 40-80 Nt/Sec

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RNA Polymerase Molecules Proceeding at a Maximal Speed of About 40-80 Nt/Sec Microbial Regulatory Systems • Regulatory system couples growth with available resources • Some proteins and RNAs are needed in the cell at about the same level under all growth conditions: constitutive expression Ribosomal Binding Site • 2 major approaches to regulate protein function: A. Control protein amount B. Control protein activity • Amount of protein synthesized can be regulated at either the level of transcription, by varying the amount of mRNA made, at the level of translation, by translating or not translating the mRNA—> gene expression etal. 2020 Madigan • After the protein has been synthesized, post- translational regulatory processes 1 Transcription of RNA in E. coli of both mRNA and the stable rRNA and tRNA, is carried out by ≈1000-10,000 RNA polymerase molecules proceeding at a maximal speed of about 40-80 nt/sec Translation of proteins in E. coli is carried out by ≈10,000-100,000 ribosomes and proceeds at a maximal speed of about 20 aa/sec Electron microscopy image of simultaneous transcription and translation. The image shows bacterial DNA and its associated mRNA transcripts, each of which is occupied by ribosomes. (Adapted from O. L. Miller et al., Science 169:392, 1970) 2 RNA synthesis: Transcription_recap • RNA polymerase (multicomplex enzyme) • σ recognizes the appropriate site on DNA for transcription to begin (σ dissociates from holoenzyme once a short sequence of RNA has been formed) • Several σ, most used σ70 • Several promoters w. 2 highly conserved regions • Upstream the transcription start site: A. 10 bases upstream, the -10 region, or 2018 Pribnow box; consensus sequence of al. TATAAT et B. 35 bases upstream consensus sequence is TTGACA, -35 region Madigan 3 RNA synthesis: Transcription_recap II • Transcription begins at a unique base just downstream from -35 and the Pribnow box • Sigma recognizes the promoter sequences on the 5′—>3′ (dark green) strand of DNA • RNA polymerase core enzyme will 2018 al. actually transcribe the light green et strand (that runs 3′—>5′) b/c core enzyme synthesizes 5′—>3′ Madigan direction 4 DNA-Protein Interaction: Madigan etal. 2020 Madigan Regulation • For a gene to be transcribed: RNA pol & σ must recognize a specific promoter site on the DNA • Regulatory proteins influence protein binding to specific DNA sites —> gene expression by turning transcription on or off • Protein-nucleic acid interactions are central to replication, transcription, translation, their regulation • Protein–nucleic acid interactions may be specific or nonspecific • DNA-binding proteins specificity: interactions side chains aa - chemical groups on nitrogenous bases & sugar • DNA major groove is the main site of protein binding —> inverted repeat • DNA-binding proteins are often homodimeric, 2 identical polypeptide subunits w. domains (= regions of the protein with a specific structure and function) • DNA-binding proteins are enzymes catalyzing a specific reaction on the DNA (e.g. transcription) • Binding event either blocks transcription (- regulation) or activates it (+ regulation) • Regulation by factors that bind directly to the RNA polymerase is complemented by factors that directly target the promoter DNA: Transcription Factors 5 Repression or activation at promoters by transcription factors , 2016 Browning & Busby Transcription factors known as repressors and activators use one of several mechanisms to repress and activate transcription initiation, respectively: RNA polymerase holoenzyme that contains σ70 in E. coli 6 Control Architecture • Genes encoding the enzymes that function in steps of the same biochemical pathway are sometimes clustered groups is called an operon • An operon is transcribed to form a single mRNA that encodes several different proteins and is regulated as a unit (cluster of genes) • E. coli has promoters under positive control and negative control • Operons have promoters with multiple types of control and some have more than one promoter, each with its own control system • Diverse localization of operons • When more than one operon is under the control of a single regulatory protein, these operons are collectively called a regulon • Arg biosynthetic enzymes are encoded by the Arg regulon Madigan et al. 2020 7 Global Networks • Global control systems regulate many genes comprising more than one regulon • Global control networks may include activators, repressors, signal molecules, two- component regulatory systems, regulatory RNA & alternative sigma factors Madigan et al. 2020 8 Promoters under positive or negative control • Repressor’s role (protein) is to stop transcription, regulation by repressors is called negative control • In positive control of transcription, the regulatory protein is an activator that activates the binding of RNA polymerase to DNA • Promoter sequences are DNA sequences that define where transcription of a gene by RNA polymerase begins: a.10 bases upstream, the -10 region, or Pribnow box; consensus sequence of TATAAT; b. 35 bases upstream consensus sequence is TTGACA, -35 region • Enzymes catalyzing synthesis of a specific product are not made if the product is already present in the medium in sufficient amounts • Enzyme repression: typically affects biosynthetic (anabolic) enzymes • Enzyme induction: enzyme is made only when substrate is present affects degradative (catabolic) enzymes • Operator: biding site of Repressor 9 Negative vs Positive Control • Repressor’s role is to stop transcription, regulation by repressors is called negative control • DNA-binding proteins vary in concentration and affinity and thus control is quantitative • When a gene is “fully repressed” there is often a very low level of basal transcription • In positive control of transcription, the Madigan et al. 2018 regulatory protein is an activator that activates the binding of RNA polymerase to DNA • Enzymes for maltose (disaccaride) catabolism in E. coli are synthesized only after the addition of • DNA region is activator-binding site maltose to the medium • RNA polymerase has difficulty binding • Maltose activator protein cannot bind to the DNA promoters unless it first binds maltose (inducer) • Activator proteins help RNA polymerase to recognize promoter • When maltose activator protein binds to DNA, it allows RNA polymerase to begin transcription 10 Negative Control: Induction • Utilization of sugar lactose as a carbon and energy source by E. coli, the enzymes for which are encoded by the lac operon • Induction of b-galactosidase, the enzyme that cleaves lactose into glucose and galactose • Synthesis begins almost immediately after lactose is added • 3 genes in lac operon encode 3 proteins, are induced simultaneously • A substance that induces enzyme synthesis is called an inducer Madigan et al. 2020 • Repressor protein is active in the absence of the inducer, completely blocking transcription • Isopropylthiogalactoside (IPTG), for instance, is an inducer of b-galactosidase even though IPTG cannot be hydrolyzed by this enzyme https://youtu.be/10YWgqmAEsQ 11 Negative Control: Repression • In E. coli enzymes for Arg synthesis are made only when Arg is absent—> an excess of Arg decreases the synthesis of these enzymes: enzyme repression • Final product of a particular biosynthetic pathway represses the enzymes of the pathway —> organism does not waste energy and nutrients synthesizing unneeded enzymes • A substance that represses enzyme synthesis is called a corepressor, Arg (effector) • Repressor protein is allosteric —> its conformation is altered when effector binds to it • By binding its effector, repressor protein is activated —> bind to a specific region Operator (near the promoter of the gene) Madigan et al. 2020 12 Global Control System • An organism needs to regulate many unrelated genes simultaneously in response to a change in its environment • Global control systems: regulatory mechanisms responding to environmental signals by regulating the transcription of many different genes • In a complex environment, the presence of a favored carbon source represses the induction of pathways that catabolize other carbon sources • Catabolite repression ensures that the organism uses the best carbon and energy source first (e.g. glucose) • Catabolic operons: lac, malt, genes for the synthesis of flagella (bc if bacteria have a good carbon source available, no need to swim around) • One consequence of catabolite repression —> 2 exponential growth phases: diauxic growth Madigan etal. 2020 Madigan • If two usable energy sources are available, the cells first consume the better energy source 13 Catabolite repression, I • Catabolite repression relies on an activator protein (positive control): cyclic AMP receptor protein (CRP) a dimer • A gene that encodes a catabolite-repressible enzyme is expressed only if CRP binds to DNA promoter region —> allowing RNA polymerase binding to promoter • Effector is cAMP derived from a nucleic acid precursor, it is a regulatory nucleotide • Cyclic di-GMP (biofilm formation) • Guanosine tetraphosphate (ppGpp, stringent response) • Cyclic AMP is synthesized from ATP by an enzyme called adenylate cyclase • Glucose inhibits cyclic AMP synthesis and stimulates cyclic AMP transport out of the cell Madigan et al. 2020 • Direct cause of catabolite repression is low level of cyclic AMP 14 Catabolite repression, II For lac genes to be transcribed: (1) Level of cyclic AMP must be high enough for the CRP protein to bind to the CRP-binding site (positive control) (2) Lactose or another suitable inducer must be present so that the lactose repressor (LacI protein)
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