Monday Oct 2 - Ch 7 -Brock - MIT OpenCourseWare

Systems MicrobiologyMonday Oct 2 - Ch 7 -BrockInformation flow in biological systems DNA replication Transcription Translation

Reverse TranscriptionCentral DogmaDNAReplicationTranscriptionImages removed due tocopyright restrictions.RNATranslationProtein

Flow of informationtranscriptiontranslationreplicationDNA DNA RNA protein

ring numberingsystem fordeoxyribose5’-C1’4’5' end-P-O-COO-3’ endOPO3’2’CHOssDNA

In a nucleotide, e.g., adenosine monophosphate (AMP), the base isbonded to a ribose sugar, which has a phosphate in ester linkage tothe 5' hydroxyl.NH2NH2NNNNNHO3PriboseHOOCH2H 1'H3'OHHOH2'adenosineNN 25' CH2NNNN4'adenineadenineNHONH2HOHHOHHOHadenosine monophosphate (AMP)

Nucleic acids have abackbone ofalternating Pi &ribose moieties.Phosphodiesterlinkages form as the5' phosphate of onenucleotide forms anester link with the 3'OH of the adjacentnucleotide.NH2adenineNN5' end O O POO5'CH24'H OOHHOHOPH 1'riboseN2'3'OcytosineNO5'CH2OOHH Onucleic acidNH2NN HHOH3'OPOriboseO(etc)3' end

HN HCytosineNNHNNNGuanineNOBackboneOH NHydrogenbondBackboneHHOCH3ThymineH NN HNONHydrogenbondNN AdenineNBackboneBackboneFigure by MIT OCW.

Diagram of genetic structure removed due to copyright restrictions.See Figure 7-4 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms. 11th ed.Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

Replication

DNA Replication A fundamental process Experimentally demonstratedDNADNADNA

DNA ReplicationTHREE HYPOTHESES FOR DNA REPLICATION

Stable isoptopes in biologySEPARATION OF DNAS BY CESIUM CHLORIDE DENSITY GRADIENT CENTRIFUGATION(a) Photo of DNA in ultracentrifuge rotor made with UV light(b) Densitometric trace of UV scan

D L 151415N labeled1. Grow E coli so DNA uniformlyN labeledN labeled142. AddN labeled to growth media and observe resultover several generations of growthPREDICTED DENSITIES OFNEWLY REPLICATED DNAMOLECULES ACCORDINGTO THE THREE HYPOTHESESABOUT DNA REPLICATION

Image of experimental results removed due to copyright restrictions.See Meselson, and Stahl. "The Replication of DNA in Escherichia coli."PNAS 44 (1958): 674, f. 4.RESULTS OF CsCl GRADIENTULTRACENTRIFUGATIONEXPERIMENT SHOWINGDISTRIBUTION OF DNADENSITY IN E. coli CELLSAFTER 0 TO 4.1GENERATIONS OF GROWTHTHIS EXPERIMENT ESTABLISHEDTHAT DNA REPLICATION ISSEMICONSERVATIVE

Conclusion1. DNA replication is semi-conservative

DNA Replication ProcessDNADNADNADiagram removed due to copyright restrictions.See Figure 7-12 in Madigan, Michael, and John Martinko. BrockBiology of Microorganisms. 11th ed. Upper Saddle River,NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

All DNA polymerases require a primerDNA is synthesized 5' to 3'RNA primerDNAPPP-5'3'-OH5'3'DNAFigure by MIT OCW.PRIMING OF DNA SYNTHESIS BYSHORT SEQUENCES OF RNA (RED)DNA POLYMERASE USES THEPRIMERS AS STARTING POINTSTO SYNTHESIZE PROGENYDNA STRANDS (GREEN ARROWS)

Table of the major enzymes involved in DNA replication in bacteria removed due to copyright restrictions.See Table 7-3 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

Helicase & topoisomeraseUnwind & remove supercoils in duplex DNA

ssDNA binding proteinbinds to and stabilizes ssDNAprevents base pairingssDNA binding protein

primasesynthesizes a short RNA primerusing a DNA templateprimaseRNA primer(a short starting sequencemade of RNA)

DNA polymerase IIISynthesizes DNA 5’- 3’, bypriming off the RNA primeron the lagging strand template.Also has 3’- 5’ proofreading activity

DNA polymerase ISynthesizes DNA from a DNAtemplate and alsoremoves RNA primers from the“Okazaki fragments”.

DNA ligaseJoins DNA strands together byforming phosphodiester bondsDNA ligase

replication fork5'lagging strand3'5'leading strandtemplate strands3'

Leading strandsynthesis5'RNA primerhelicasessDNA binding proteins3'

5'DNA polymerasehelicasessDNA binding proteins3'

Leading strand synthesis5'DNA pol IIIhelicaseDNAssDNA binding proteins3'

ProofreadingPol III removes misincorporated bases using 3' to 5'exonuclease activityThis decreases the error rate to about 10-10 per basepair insertedDiagram of DNA proofreading removed due to copyright restrictions.See Figure 7-20 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

Lagging strand synthesis(discontinuous)Okazakifragment3'5'( 1000 bases)(primase)helicasessDNA binding proteinspol III3'

Primer removalpolIII3'5'pol Ipol I5’ to 3’exonucleaseactivity

LigationDNA ligase

5'RNA primerLagging strandPrimase3'5'Single-strand binding proteinHelicaseFree 3'-OHDNA polymerase IIILeading strandRNA primer3'5'Figure by MIT OCW.

DNA SYNTHESIS HAPPEN BIDIRECTIONALLY, FROM INITIATION SITE“REPLICATION BUBBLE”

Origin of replicationReplication forksThetaStructureNewly synthesizedDNAFigure by MIT OCW.

Flow of informationreplicationDNA DNAtranscription RNAtranslation protein

Regulatory pathways in prokaryotesRegulateenzyme activitySubstrateProductRegulate enzyme synthesisAt translationNo ProductEnzyme AEnzyme BAt transcriptionNo EnzymeTranslationNo mRNATranscriptionGene AGene BGene CGene DFigure by MIT OCW.

Prokaryotic transcriptionTranscribed regionsRNA polymerasePromotersTerminatorsSigma factor

5' end-P-O-COO-3’ endHOOPURACILOCHOHOssDNA

TranscriptionDiagram of RNA transcription removed due to copyright restrictions.See Figure 7-29a in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

DNA dependent RNA Polymerase (RNAP)recognizes promoter sequence and initiatestranscriptionTerminateInitiatePromoter region{3'3'5'{5'1 GeneSigma aids in recognition of promoterand initiation site5'3'3'5'Sigma factorRNA polymerase(core enzyme)Figure by MIT OCW.

Synthesis of the mRNA transcript (5’ 3’)complementary to one of the twp strands – thetemplate strand – sigma dissociatesElongation PhaseSigma3'5'3'5'5'Transcription begins;sigma released5'3'3'5'RNA chain growth5'Figure by MIT OCW.

Elongation phase continues until the RNAP reaches aterminator and dissociates5'3'3'5'Termination site reached;chain growth stops5'3'5'3'3'Release of polymeraseand RNA5'5'Figure by MIT OCW.

Initiation of transcription begins with promoter binding byRNAP holoenzymeholoenzyme RNAP core SigmaDiagram of RNA polymerase and transcription removed due to copyright restrictions.

Architecture of a vegetative (σ70) promoter-core promoter recognized by sigma factorT T G A C AT A T A A T69 79 61 56 54 5477 76 60 61 56 8217 bp spacer (43)% occurenceA47-60UP Element-35-10Core Promoter 1DSR

Alternative sigma factors bind to core RNA pol and direct it todifferent promoters.E. coli RNA pol holoenzyme is α2ββ’σSigma 70 is used for ‘normal’ promotersSigma 32 is used for heat-shock promotersSigma 54 is used for N limitation promotersGenerpoDrpoHrpoNSigma factorσ70σ32σ54-35SpacingTTGACA16-18 bpCCCTTGAA 13-15 bpCTGGNA6 bp–10TATAATCCCGATNTTTGCA

What dictates the transcriptional activity of a gene?Promoter strengthHow similar are the promoter core elements (-10,-35, andtheir spacing) to the consensus?- in general the more similar they are, the more activethe promoter will be to initiate transcription- however, some positions are more importantthan othersTTGACA T---AStrong promoterTCAGTT---19bp---GATAAC---AWeaker promoter

Non-core sequences can affect promoter strength1. Extended –10 sequencessome promoters have longer –10 elements2. UP elementsother promoters have AT rich sequences just upstream of the–35 that elevate transcription rate3. Downstream elementssequences immediately downstream of the start site canaffect the overall efficiency of transcription initiation

Initiation complexes through elongationDiagram removed due to copyright restrictions.Karp, 1999, Molecular Cell Biology, Wiley and SonsRNAP promoterRNAPRNAPClosed complexOpen complexRNAPElongation complex

The transcription cycle- can be viewed as acycle1. InitiationDiagram removed due to copyright restrictions.2. Elongation3. TerminationMooney and Landik, 1999. Cell98: 687

Mechanism of transcriptional initiationNTPsk1R PRPCk-1Described by aequilibrium constantcalled KIk2k-2RPOk3RPIRPEk-3abortivetranscriptsKI RPC/(R P)R – RNAPP – PromoterRPC – closed complexRPO – open complexRPI – initiation complexRPE- elongation complexk4The rate of opencomplex formation isoften called kIIThis transitionis called“promoterclearance”described by kIV

Transcription terminationDiagram of transcription termination removed due to copyright restrictions.See Figure 7-32 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms. 11th ed.Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

Some differences between eukaryotic &prokaryotic transcription.Eukaryotic mRNAs are usuallyspliced,capped and tailed, in the nucleus.Eukaryotes do NOT have classical operons.RNA polymerase structure/function differInitiation complexes differ Sigma factor vs. TBPProkaryotic genes very very rarely have introns

DRUGS THAT INHIBIT TRANSCRIPTION &/or DNA REPLICATIONANTIBIOTICActinomycin DAdriamycin.HClAphidicolinTARGET; MODE OF ACTIONTranscription; inhibits DNA-dependent RNA synthesis by binding DNADNA replication & Transcription; Inhibits DNA and RNA synthesis by binding DNADNA replication ; Inhibits alpha-type polymerase (eukaryotic and viral)Bleomycin.sulfateDNA replication ; reacts with DNA and causes chain breakChromomycin A3Transcription ; Inhibitor of DNA-dependent RNA-synthesisMithramycin ATranscription ; inhibits RNA synthesis by complexing with DNAMitomycin CDNA replication ; Anti-tumor antibiotic. Binds covalently to DNANalidixic acidDNA replication; Inhibitor of bacterial DNA gyrase (a topisomerase inhibitor)Netropsin.DNA replication; Peptide antibiotic. Binds to AT-rich regions in the minor groove of DNANovobiocinDNA replication; Inhibitor of bacterial DNA gyraseRifampicinTranscription ; Inhibitor of DNA-dependent RNA-polymerasNovobiocin.DNA replication; Causes DNA methylation and DNA strand breaks

TranslationCoupled script processingDiagram of transcription and translation in prokaryotes vs. eukaryotes removed due to coypyright restrictions.

Coupled transcription/translationMicroscopic photographs of transcription and translation removed due to copyright restrictions.

Flow of informationreplicationDNA DNAtranscription RNAtranslation protein

Overview of prokaryotic translationProtein synthesis from an mRNA template.translated regionmRNAphetranslationprotein of specific amino acid sequence

Key components of translationMessenger RNATransfer RNAribosomes and rRNA

Simple structure of a prokaryotic geneσ70-type PromoterTranscriptional terminatorUUUUUUUTTGACA orysequences1-500 nts2-8 nts( 5 nts)Ribosome Binding Site (RBS)Stopcoding sequencevariable

Shine-Dalgarno sequence AGGAGG, ribosome binding sequence,critical for ribosome bindingStart codonsAUG, GUG, or UUGStop codons (nonsense codons)UAA, UGA, or UAG

Secondary Structure: small subunit ribosomal RNA5’mRNA AGGAGGU 3’3’rRNA UCCUCCA 5’Shine Delgarno seq.Blue Universal sitesCourtesy of the Comparative RNA Web Site. Used with permission.

THE GENETIC CODE Series of codons that determinesthe amino acid sequence of theencoded protein. Coding sequences have an averageof about 300 codons. Except for the stop codon, eachcodon specifies a particular aminoacid.

The genetic codeTable of the genetic code removed due to copyright restrictions.See Table 7-5 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms. 11th ed.Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

The genetic code is degenerate.more than one codon can codefor the same amino acidUUU phenylalanineUUC phenylalanine

SynonymsDifferent codons can code for the same amino acidUUU phenylalanineUUC phenylalanineNot all synonyms are used with equalfrequency. This is called "codon usagebias".