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Chapter 29

Notes Chapter 29 (Lectures 13-17) - Biochem 2B03.docx

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McMaster University
Margaret Fahnestock

Biochem 2B03 Jasmyn Lee Part 4: Information Transfer Chapter 29: Transcription and the Regulation of Gene Expression 29.1 How Are Genes Transcribed in Prokaryotes? o RNA is synthesized by a single species of DNA-dependent RNA polymerase, with the exception of the short RNA primers formed by primase during DNA replication o RNA polymerase links ribonucleoside 5’-triphosphates (NTP = ATP, GTP, CTP, UTP) in an order specified by base pairing with a DNA template o n NTP  (NMP) + n PP i o RNA pol moves along DNA strand in the 3’5’ direction, joining the 5’-phosphate of an incoming ribonucleotide to the 3’-OH of the previous residue o Subsequent hydrolysis of PPito inorganic phosphate by the pyrophosphatases present in all cells removes the product Pi , making the polymerase reaction thermodynamically favourable o Note: o mRNA synthesis proceeds 5’ to 3’ – chemistry is identical to DNA synthesis o Protein synthesis proceeds N-terminus to C-terminus Prokaryotic RNA Polymerases Use Their Sigma Subunits to Identify Sites Where Transcription Begins Action of RNA polymerase  Nucleophilic attack of 3’-OH of preceding nucleotide on the α-phosphate of the incoming nucleoside triphosphate  Pyrophosphate is released and hydrolyzed by inorganic pyrophosphatase Chemical reaction of RNA synthesis  Chain Initiation o pppN1 + pppN2  pppN1pN2 + pp i  Chain Elongation o pppN1pN2 + pppN3  pppN1pN2pN3 + pp i  pppN = ATP, CTP, GTP, and UTP as determined by the DNA template One RNAP in prokaryotes  E. coli RNAP found in at least two forms: holoenzyme and core enzyme  α ββ’σ holoenzyme – promoter recognition and initiation 2  α2ββ’ core enzyme – RNA synthesis but cant initiate (cannot recognize promoters)  β and β’ form a catalytic claw that DNA passes through  β’ binds DNA  β binds NTPs and interacts with σ  σ recognizes promoters (nucleotide sequences that identify the location of transcription start Structure of the core RNA site)  σ subunits assembly and for activation of RNAP by regulatory proteins polymerase from Thermus thermophiles The Process of Transcription Has Four Stages 1. Promoter recognition – binding of RNA polymerase holoenzyme at promoter site 2. Chain initiation (first bond synthesis) – initiation of polymerization 3. Chain elongation 4. Chain termination 1 Biochem 2B03 Jasmyn Lee Events in Initiation and Elongation  Step 1: σ factor of holoenzyme RNAP recognizes promoter sequences  Step 2: formation of RNAP-closed promoter complex o dsDNA has no yet been unwound so that the RNAP can read the base sequences of the DNA template strand and transcribe it into a complementary RNA sequence  Step 3: DNA unwinding by RNAP and formation of open promoter complex  Step 4: RNAP initiates RNA synthesis at a purine nucleotide site  Step 5: RNAP adds ~4 more nucleotides  Step 6: σ falls off and core enzyme continue the synthesis Binding of RNAP to template DNA  RNAP holoenzyme binds nonspecifically to DNA with low affinity and migrates along it looking for promoter  Sigma subunit recognizes promoter sequence -6 -9  RNAP holoenzyme and promoter form “closed promoter complex” (DNA in “closed” conformation) – K d 10 to 10 M  RNAP unwinds about 12 pairs to form “open promoter complex” – Kd= 10 -1M How does an RNAP find a promoter? Properties of Promoters 2 Biochem 2B03 Jasmyn Lee  RNAP binding typically protects a nucleotide sequence spanning the region from -70 to +20  +1 = transcription start site (there is no 0) – base in the nontemplate strand is identical with the RNA transcript  Nontemplate strand bases in the 5’ direction = minus direction  lie “upstream” of transcription start site  Nontemplate strand bases in the 3’ direction = plus direction  lie “downstream” of transcription start site  Segment of promoter in lac operon a) E. Coli promoters are highly conserved b) Promoters recognized by the principal σ factor (σ ) serve as the paradigm for prokaryotic promoters a. Promoters vary in size from 200-200 bp; typically ~40 bp region b. Typically located on the 5’-side of the transcription start site c) Two consensus sequence elements within the promoter, separated by ~17 bp of nonconserved sequence a. Consensus sequence – the bases that appear with highest frequency at each position when a series of sequences believed to have common function is compared b. Pribnow Box near -10; consensus sequence is the hexameric TATAAT c. Sequence in the -35 region; consensus sequence is the hexameric TTGACA d) RNAP uses σ-subunit to bind to conserved sequences a. The more closely the -35 region sequence corresponds to its consensus sequence, the greater is the efficiency of transcription of the gene e) Consensus compiled from 298 E. coli promoters f) Pribnow box is ideal for unwinding Initiation of Transcription  RNAP has two binding sites for NTPs; initiation site and elongation site  Initiation site – binds the first NTP within the open promoter complex; prefers to bind ATP and GTP (most RNAs begin with a purine at 5’ end)  Elongation site – binds the second NTP  3’-OH of first NTP attacks alpha-P (α-phosphorous atom) of the second NTP to form a new phosphoester bond (eliminating PPi)  When RNA chain grows to 6-10 units, σ subunit dissociates and the core enzyme clears the promoter, completing “initiation”  Core enzyme continues to synthesize the remainder of the mRNA  The DNA duplex is unwound just ahead of the core RNAP as it advances the 3’-end of the RNA chain  Open Complex Formation o RNAP contacts promoter around ~35 and ~10 regions – these sequences bound by the σ factor Chain Elongation 3 Biochem 2B03 Jasmyn Lee  RNAP core is the elongation enzyme  RNAP is fairly accurate – error rate: 1/10,000 bases o Acceptable error rate – many transcripts are made from each gene and most transcripts are smaller than 10 kb  Elongation rate is 20-50 bases/sec – slower in G/C rich regions and faster everywhere else  Topoisomerases precede and follow polymerase to relieve supercoiling  Supercoiling versus Transcription o DNA is supercoiled to avoid tangling of nucleic acid strands if RNA wrapped around DNA as RNA polymerase followed template strand around axis of DNA duplex o Positive supercoils formed ahead of transcription bubble o Negative supercoils formed behind transcription bubble o During transcription, supercoiling is regulated by topoisomerases o Gyrase – ahead of the transcription bubble, removes positive supercoils introduced by the forward movement of RNAP (therefore introduces negative supercoils) o Topoisomerase I behind the transcription bubble removes negative supercoils, maintaining balance Chain Termination  Two mechanisms of transcription termination mechanisms – factor dependent and factor independent 1. Factor independent of intrinsic terminators o Terminators (termination sites) encoded in the DNA – no factor requirement o Some DNA sequences cause spontaneous termination of RNA transcription o The transcript forms very stable hairpin structure via intrachain base pairing due to inverted repeats (typically G:C rich) that are separated by a non-repeating segment  Molecular Mechanism 2. Factor dependent or Rho-dependent o Dependent on a specific transcription termination factor (protein) Rho o Rho is a hexameric ATP-dependent RNA/DNA helicase that catalyzes the unwinding of RNA:DNA hybrid duplexes o Binds to transcript (that are unoccupied by translating ribosomes) and migrates along it (5’3’) until it reaches transcription bubble o When it reaches the RNA polymerase active site it unwinds the RNA/DNA duplex and releases the RNA chain o Characteristics of Rho-dependent terminators  Pause site for RNA polymerase  Upstream mRNA – no coding sequence  Upstream mRNA – no “secondary structure”  Rich in C residues, low in G residues 4 Biochem 2B03 Jasmyn Lee  Rho attaches to a recognition site (C-rich region) on mRNA  Rho moves along mRNA behind RNAP  When RNAP pauses at termination site, rho unwinds the DNA:RNA hybrid  The nascent mRNA is released 29.2 How Is Transcription Regulated in Prokaryotes?  Genes encoding enzymes of a metabolic pathway are grouped as operons (adjacent to one another in a cluster on a chromosome) o Allows coordinated transcription onto a single polycistronic mRNA  Polycistronic mRNA – single RNA transcript that encodes more than one polypeptide  The operator adjacent to such a unit determines whether the operon is transcribed o Located next to the promoter  Regulatory proteins work with operators to control transcription of gene cluster by controlling access of RNA polymerase to the promoter  Lac operon – regulation of initiation by repression  Amino acid biosynthetic operons – regulation by attenuation  Positive regulation of initiation (activation)  Complex promoters – araBAD Transcription of Operons Is Controlled By Induction and Repression  In prokaryotes; gene expression is often responsive to small molecules serving as signals of the nutritional or environmental conditions confronting the cell  Induction – increased synthesis of enzymes in response to the presence of a particular substrate o Co-inducers/inducers – substrates capable of activating synthesis of the enzymes that metabolize them o Gratuitous Inducers – substrate analogs that can induce enzyme synthesis even though the enzymes are incapable of metabolizing them o Eg/  Lactose can serve as both carbon and energy source for E. coli  Metabolism of lactose depends on hydrolysis into its component sugars (glucose and galactose) by β- galatosidase  Absence of lactose – E. coli cells contain little β-galactosidase  Lactose availability induces the synthesis of β-galactosidase by activating transcription of the lac operon  LacZ (gene in lac operon) – structural gene for β-galactosidase  Repression – decreased synthesis of enzymes in response to a specific metabolite o Co-repressor – metabolite depresses synthesis of its own biosynthetic enzyme; typically an end product o Eg/ 5 Biochem 2B03 Jasmyn Lee  Enzymes of tryptophan biosynthesis in E. coli are encoded in the trp operon  If sufficient Trp is available to the growing bacterial culture, the trp operon is not transcribed – Trp biosynthetic enzymes are not made The lac Operon Serves as a Paradigm of Operons  Operon Hypothesis – accounts for the coordinated regulation of related metabolic enzymes o 1961; Francois Jacob and Jacques Monod  Two classes of genes – distinguished by mutation o Structural Genes – for the enzymes  Mutation would abolish one particular enzymatic activity o Regulatory Genes – control expression of the structural genes  Mutation would affect all other enzymes under its control  Eg/ Mutations were known in E. coli for lactose metabolism o Bacteria with mutations in either the lacZ gene or the lacY gene could no longer metabolize lactose -  lacZ mutations (lacZ strains) – β-galactosidase activity was absent  Therefore – encodes β-galactosidase -  lacY mutations (lacY strains) – Lactose was no longer transported into the cell  Therefore – β-galactosidase permease structural gene; active in β-galactosidase transport into the cell -  lacI mutations (lacI strains) – expressed β-galactosidase activity and immediately transported lactose, without prior exposure to an induced; single mutation led to the expression of lactose metabolic functions independent of the inducer  Constitutive Expression – expression of genes independently of regulation  Therefore – properties of a regulatory gene Lac Repressor Is a Negative Regulator of the lac Operon The Mode of Action of Lac Repressor  Negative Regulation – transcription is on unless turned off by a repressor  Structural genes of the lac operon are controlled by negative regulation o Transcribed to give an mRNA unless turned off by the lacI gene product (lac repressor; protein)  Lac repressor – negative regulator of the lac operon o Has N-terminal DNA binding domain o Rest of protein function sin inducer binding and tetramer formation  Basis for regulation by Lac repressor is “affinity and specificity”  Absence of Inducer o lac repressor blocks lac gene expression by binding to the operator DNA site upstream from the lac structural gene o lac operator – palindromic DNA sequence o RNAP can initiate transcription at the promoter even in the presence of a lac repressor, but the lac repressor blocks elongation of transcription so initiation is aborted  lacI mutations – lac repressor is absent or defective in binding to operator DNA, lac gene transcription is not blocked and the lac operon is constitutively expressed in these mutations  Induction o lac repressor tetramer has four inducer binding sites – when appropriate β-galactosides occupy inducer site, a conformational change occurs  Causes inducer:lac repressor complex to dissociates from DNA  RNA polymerase transcribes the structural genes o Reverses rapidly – once inducer is used up through metabolism by the enzymes, free lac repressor reassociates with the operator DNA, transcription is halted, residual lac mRNA is degraded  Absence of Inducer o lac repressor binds nonspecifically to  Duplex DNA with an association constant; K Af 2 x 10 M -1 13 -1  lac operator DNA sequence with much higher affinity; KA= 2 x 10 M  Lac repressor binds 10 times better to lac operator DNA than any random DNA sequence o IPTG (gratuitous inducer) binds to lac repressor; K = 10 M -1 A 10 -1  IPTG:lac repressor complex binds to operator DNA; K A 2 x 10 M  Three orders of magnitude less than the affinity of inducer-free repressor for lac operator o Lac repressor acts by binding to DNA and sliding along it, testing sequences in a one=dimensional search until it finds the lac operator; remains bound with high affinity until inducer causes this affinity to drop by 3 orders of magnitude 6 Biochem 2B03 Jasmyn Lee  Regulation of transcription elongation by attenuation  Depends on the coupling of transcription and translation The trp Operon is Regulated Through a Co-Repressor-Mediated Negative Control Circuit  Trp operon (of E. coli and S. tymphimurium) encode the five polypeptides (trp E through trpA) that assembles into the three enzymes catalyzing tryptophan synthesis from chorismate  Expression of the trp operon is under control of Trp repressor  Plentiful tryptophan o Trp repressor binds two molecules of tryptophan and associates with the trp operator that is located within the trp promoter o Trp repressor binding excludes RNA polymerase from the promoter, preventing transcription of the trp operon  Limited tryptophan o Repression is lifted – Trp repressor lacks bound Trp and has a lower affinity for the trp promoter  The behaviour of the Trp repressor corresponds to a co-repressor-mediated negative control circuit  Autogenous Regulation (Autoregulation) – regulation of gene expression by the product of the gene o Trp repressor is encoded by the trpR operon and regulates expression of the trpR operon Attenuation is a Prokaryotic Mechanism for Post-Transcriptional Regulation of Gene Expression 7 Biochem 2B03
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