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Ecclesiastes 3:1 1 To every thing there is a season, and a time to every purpose under the heaven: ©2000 Timothy G. Standish Transcription: Concentrating on Prokaryotes Timothy G. Standish, Ph. D. ©2000 Timothy G. Standish All Genes Can’t be Expressed At The Same Time Some gene products are needed by all cells all the time. These constitutive genes are expressed by all cells. Other genes are only needed by certain cells or at specific times, expression of these inducible genes is tightly controlled in most cells. For example, pancreatic b cells make insulin by expressing the insulin gene. If neurons expressed insulin, problems would result. ©2000 Timothy G. Standish Logical Expression Control Points Increasing cost DNA packaging Transcription RNA processing mRNA Export mRNA masking/unmasking and/or modification mRNA degradation Translation Protein modification Protein transport Protein degradation The logical place to control expression is before the gene is transcribed ©2000 Timothy G. Standish Introduction The Central Dogma of Molecular Biology Cell Transcription Translation Reverse tanscription DNA mRNA Ribosome Polypeptide (protein) Stages of Transcription 1. 2. 3. 4. l Transcription can be logically divided into Four distinct stages: Template recognition Initiation Elongation Termination Each stage may participate in regulation, but template recognition and termination appear to be major players ©2000 Timothy G. Standish General Model For Transcription Core Enzyme aa b s b RNA Polymerase Holoenzyme ©2000 Timothy G. Standish General Model For Transcription aa b s b ©2000 Timothy G. Standish General Model For Transcription aa b s b ©2000 Timothy G. Standish General Model For Transcription aa b s b ©2000 Timothy G. Standish General Model For Transcription s TopIsomerase I a NusAa b b Gyrase ©2000 Timothy G. Standish General Model For Transcription s a NusAa b TopIsomerase I b Gyrase Ribosome Exonuclease Ribosome ©2000 Timothy G. Standish General Model For Transcription s a NusAa b Ribosome Exonuclease b Ribosome ©2000 Timothy G. Standish General Model For Transcription s a NusAa b Exo- b Ribosome nuclease ©2000 Timothy G. Standish General Model For Transcription s a NusAa b Exo- b Ribosome nuclease ©2000 Timothy G. Standish General Model For Transcription s a NusAa b b Exonuclease ©2000 Timothy G. Standish General Model For Transcription s a NusAa b b Exonuclease ©2000 Timothy G. Standish General Model For Transcription NusA s aa b b Exonuclease ©2000 Timothy G. Standish General Model For Transcription s a NusAa b b ©2000 Timothy G. Standish RNA Polymerase 1. 2. 3. 4. 5. RNA Polymerase is a spectacular enzyme, it performs the following functions: Recognition of the promoter region Melting of DNA (Helicase + Topisomerase) RNA Priming (Primase) RNA Polymerization Recognition of terminator sequence ©2000 Timothy G. Standish Prokaryotic Transcription Initiation The s subunit of prokaryotic RNA polymerase is necessary for promoter recognition and binding of RNA polymerase to the promotor Different s subunits allow recognition of different types of promoters thus the type of genes transcribed can be modulated by altering the types of s subunits which attach to RNA polymerase ©2000 Timothy G. Standish Prokaryotic Transcription Initiation RNA Pol. s P1 Heat Shock Gene P2 Constitutive Gene Different promoters ©2000 Timothy G. Standish Prokaryotic Transcription Initiation RNA Pol. s P1 Heat Shock Gene P2 Constitutive Gene Different promoters ©2000 Timothy G. Standish Transcription Termination There are two types of termination: Rho dependent requires a protein called Rho, that binds to and slides along the RNA transcript. The terminator sequence slows down the elongation complex, Rho catches up and knocks it off the DNA Rho independent termination depends on both slowing down the elongation complex with a hairpin and a U rich region that destabilizes the elongation complex ©2000 Timothy G. Standish Termination Rho Independent RNA Pol. RNA Terminator 5’ RNA Pol. RNA 5’ ©2000 Timothy G. Standish Termination Rho Independent RNA Pol. RNA 5’ 5’ RNA Pol. RNA Terminator ©2000 Timothy G. Standish Termination Rho Dependent Terminator RNA Pol. RNA 5’ r RNA Pol. RNA 5’ r The terminator sequence slows RNA polymerase ©2000 Timothy G. Standish Termination Rho Dependent Terminator RNA Pol. RNA 5’ r Help, rho hit me! r RNA 5’ RNA Pol. Rho catches up with RNA polymerase ©2000 Timothy G. Standish Termination Rho Dependent Terminator RNA Pol. RNA 5’ 5’ r The elongation complex disintegrates RNA Pol. RNA ©2000 Timothy G. Standish ©2000 Timothy G. Standish Transcription Coding (sense) strand 5’ 3’ 3’ 5’ Template (antisense) strand ©2000 Timothy G. Standish Transcription Coding (sense) strand 5’ 3’ 3’ 5’ 5’ RNA Pol. Template (antisense) strand RNA ©2000 Timothy G. Standish Transcription Coding (sense) strand 5’ 3’ 3’ 5’ Template (antisense) strand RNA Pol. 5’ ©2000 Timothy G. Standish Products of Transcription Transcription produces three major RNA products: 1 Ribosomal RNA (rRNA) - Several rRNAs are vital constituents of ribosomes 2 Transfer RNA (tRNA) - The molecule that physically couples nucleic acid codons with specific amino acids 3 Messenger RNA (mRNA) - The nucleic acid messenger that carries encoded information from genes on DNA to the protein manufacturing ribosomes ©2000 Timothy G. Standish Transfer RNA (tRNA) Acts as the adapter molecule between the genetic code on mRNA and the protein “language” 75-85 bases long A specific amino acid is covalently linked at the 3’ end Elsewhere on the molecule is an anticodon complimentary to the specific amino acid codon on mRNA that codes for the amino acid carried by the tRNA Contain a number of modified bases ©2000 Timothy G. Standish A “Simple” Gene Transcription Start Site 5’ 5’ Untranslated Region 3’ Untranslated Region Protein Coding Region 3’ RNA Transcript Promoter/ Control Region Terminator Sequence ©2000 Timothy G. Standish Transcription Initiation Proteins called transcription factors bind to the promoter region of a gene If the appropriate transcription factors are present, RNA polymerase binds to form an initiation complex RNA polymerase melts the DNA at the transcription start site Polymerization of RNA begins ©2000 Timothy G. Standish Initiation T. F. Promoter T. F. RNA Pol. RNA Pol. RNA 5’ ©2000 Timothy G. Standish Transcription And Translation In Prokaryotes 5’ 3’ 3’ 5’ RNA Pol. Ribosome mRNA Ribosome 5’ ©2000 Timothy G. Standish Heat Shock Response Elements Sudden changes in the temperature of cells cause stress in response to which heat shock genes are expressed At least some heat shock genes are thought to be chaperones that help proteins fold correctly Heat shock genes have Heat Shock Elements (HSEs) in their control regions Heat Shock Transcription Factors (HSTFs) bind the HSEs up regulating expression of heat shock gene products ©2000 Timothy G. Standish ©2000 Timothy G. Standish Control of Gene Expression Cytoplasm Packaging Degradation DNA Transcription Transportation Modification RNA RNA Processing mRNA G G AAAAAA Nucleus Export Degradation etc. AAAAAA Translation ©2000 Timothy G. Standish A “Simple” Gene Transcription Start Site 5’ Untranslated Region 5’ 3’ Untranslated Region Protein Coding Region 3’ RNA Transcript Promoter/ Control Region Terminator Sequence ©2000 Timothy G. Standish Initiation T. F. Promoter T. F. RNA Pol. RNA Pol. T. F. RNA 5’ ©2000 Timothy G. Standish Transcription And Translation In Prokaryotes 5’ 3’ 3’ 5’ RNA Pol. Ribosome mRNA Ribosome 5’ ©2000 Timothy G. Standish