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Lecture 13

Chapter 29 (Lecture 13-17) – Review Quicksheet.docx

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Margaret Fahnestock

Chapter 29 – Review Quicksheet Biochem 2B03 Prokaryotes Transcription RNAP  Regulatory proteins assemble σ subunits and activate RNAP  Core enzyme α β2’ 1. Promoter Recognition  Holoenzyme αββ’σ -8 -9 o σ recognizes promoters  RNAP binds non-specifically to DNA (low affinitd; K = 10 – 10 M)  σ (of RNAP holoenzyme) recognizes promoter sequence  forms closed promoter sequence (K d 10 -14M)  β’ binds DNA  Promoters are highly conserved; consensus sequences  β binds NTP and interacts o Pribnow Box at -10 TATAAT with σ o Sequence near -35 TCTTGACAT 2. Chain Initiation (first bond synthesis)  RNAP unwinds DNA  forms open promoter complex (K = 1d -1M)  At initiation site RNAP initiates RNA synthesis; binds first NTP within open promoter sequence (prefers ATP and GTP; purines) st  Nuc attack of 3’-OH of 1 NTP on α-P of incoming NTP  pyrophosphate released and hydrolyzed by inorganic pyrophosphatase o pppN1 + pppN2  pppN1pN2 + pp i (pppN = ATP, CTP, GTP, UTP as determined by DNA template)  RNA grows 6-10 units; σ falls off 3. Chain Elongation  RNAP core continues synthesis (elongation rate 20-50 bases/second; fairly accurate – error rate 1/10 000 bases) o pppN1pN2 + pppN3  pppN1pN2pN3 + pp i (pppN = ATP, CTP, GTP, UTP as determined by DNA template)  Supercoils form – negative behind, positive ahead of transcription bubble  Gyrase ahead of transcription bubble removes positive transcription bubbles (introduces negative supercoils)  Topoisomerase I behind transcription bubble removes negative supercoils – maintains balance 4. Chain Termination  Factor Independent of Intrinsic Terminators o Terminators (termination sites) encoded in DNA – cause spontaneous termination of RNA transcription o Transcript forms stable hairpin structure (intrachain base pairing, G:C rich)  RNAP pauses and undergoes conformational change – transcription stops  Factor Dependent (Rho Dependent) o Rho – hexameric ATP-dependent RNA/DNA helicase – unwinds RNA:DNA duplex o Rho attaches to recognition site (C-rich region) and migrates along transcript (5’-3’)  Upstream mRNA – no coding sequence or secondary structure o Rho-Dependent Terminators (termination sequence) causes RNAP to pause o Rho reaches transcription bubble – unwinds RNA/DNA duplexand releases RNA chain Transcription Regulation  Operon – group of genes encoding enzymes of a metabolic pathway; operator, promoter and one or more structural genes o Structural genes – determines the amino acid sequence of a protein o Operator (O) – DNA site that regulates activity of structural genes by interacting with repressor o Promoter (P) – DNA sequence to which RNA polymerase bindsto initiate mRNA transcription  Constitutive Protein’s – synthesized at a constant rate (not regulated)  Inducible Proteins – synthesized at variable rates dependent on cells circumstances (regulated); Negative Control/Regulation – transcription is on unless turned off (by repressor)  Inducer inactivates repressor – inducer present = transcription  lac Operon o When glucose (preferred carbon source) is not present – cell can use lactose; must be hydrolyzed by β-galactosidase o Lactose Absent (glucose present) – little β-galactosidase; repressor binds to operator and prevents transcription o Lactose Present (low glucose) – abundant β-galactosidase; inducer binds to repressor, repressor cannot bind to operator, transcription occurs  trp Operon o Attenuation – regulates transcription; depends on coupling transcription and translation o Leader Region – before first structural gene; encode a short leader peptide containing multiple codons for pertinent amino acid  Contains intrinsic terminator  Folding of terminator  controlled by translation of leader peptide  depends on amino acid availability o Abundant Tryptophan – termination of transcription upstream of coding sequence caused by terminator in leader region  RNAP transcribes; 1:2 hairpin is formed causing RNAP to pause elongation cycle  Ribosome begins to translate leader region; reaches RNAP – RNAP and ribosome transcribe and translate in unison  3:4 terminator hairpin forms – presence of ribosome blocks formation of 2:3 anti-terminator hairpin  Transcription of downstream genes is prevented (start codon blocked) – premature termination o Scarce Tryptophan – entire mRNA is transcribed to make tryptophan biosynthetic enzymes
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