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Exam 2

by: Hanna nune

Exam 2 Mibo 4090

Hanna nune
GPA 3.6
Prokaryotic Biology
Vincent J. Starai

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Hey Guys I made study guide for exam 2 with detailed and also some of the old exam questions and answer so check it out. Thank You
Prokaryotic Biology
Vincent J. Starai
Study Guide
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This 38 page Study Guide was uploaded by Hanna nune on Monday September 21, 2015. The Study Guide belongs to Mibo 4090 at University of Georgia taught by Vincent J. Starai in Fall 2015. Since its upload, it has received 105 views. For similar materials see Prokaryotic Biology in Microbiology at University of Georgia.


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Date Created: 09/21/15
MIBO409O Lecture 16 Replication Ch 12 Learning Objectives I Learn the activities of DnaA PolIII Poll and Tus during replication I Understand subunit structure of PolIII I Understand topoisomerases Simple dogma bears repeating I Replication DNA genomic information Transcription messenger RNA information Translationprotein synthesis Separate conserved and regulated machinery for each step Essential processes for life as we know it viruses prions protein Goal to make MORE DNA genetic information is selfish DNA review 4 deoxynucleotide bases adenine A purines guanine G thymine T pyrimidines Cytosine C Double heliX form Bases stack inside Phosphate backbone outside Base pairing review D quot H v Fry IN H ltH D m Guanine Cytosine GC base pair with 3 Hbonds Important differences for high G C organisms DNA characteristics I Usually double stranded write dsDNA I Synthesized replicated 5 to 3 molecular polarity I deoxyribose 5 phosphate I deoxyribose 3 hydroxyl I anti parallel structure strands in opposite directions semi conservative replication Semiconservative antiparallel replication ht c The MCGraw Hill Corn an 5quot 339 Parental helix Replication fork Replicas Parental New New Parental I One old plus one new strand each round Can tell difference Via adenosine methylation we ll talk more I Replication regulated by relative protein of DnaA protein 0 Low and high affinity sites at oriC origin of replication DnaA protein has multiple domains I Proteinprotein interactions DnaA DnaB II linker allowing for rotational freedom III ATPbinding domain critical for DnaADnaA binding IV DNAbinding domain standard helixtarnhelix oriC structure EDLJE a I l I a I a I g A 3 L M H I311 1 le R5122 11 HE E HE TE 94 I binds DnaA protein at multiple sites Rl R5 I R1 and R4 blue have high affinity for DnaA ADP gt orilt DnaA ATP I R2 then R35 fill in with DnaA ATP given sufficient DnaA concentration I multimerization continues to other pink sites I t unwinding DNA at I DNA unwinding Element DUE I Other DNA binding proteins involved IHF yellow Fis orange I DnaA binding to oriC is cooperative and depends on ATP DnaAloaded oriC leads to unwinding E r I i v r r 5 4 E r w of DnaAATP bound to ssDNA in unwound DNA unwinding element 0 ATP is reguired for this I The DUE is intrinsically unstable tandem repeats of 13bp ATrich regions Why is this important only one domain of DnaA seen here domain III ATPbinding region 0 termed open complex DnaAATP helps load DnaB to oriC Egan a I DnaB helicase homohexamer interacts with domain I in DnaA ATP O binds DnaC ATP first helicase loader DnaG inhibitor 0 DnaBC binds to DnaA ATP I DnaBC loads to ssDNA opposite that of DnaA ATP I Hydrolysis of ATP on DnaC fully loads DnaB removes DnaC Lagging strand DNA e1ngxaged with primase active site Lagxgwing strand DKNA bound to the pror vp osed N TD SSDMA binding site Occluwdedl leading 3 strand DNA DnaG primase synthesizes RNA primers I DNA synthesis cannot start Without a 3 OH 0 synthesis is 5 to 3 remember I RNA synthesis CAN DnaG is this polymerase 0 DNA polymerase does not care if primer is RNA or DNA just looking for free 3 OH and ssDNA I Synthesizes short ca 11 bp RNA primers as template complementary to DNA 0 after primer synthesis DnaG dissociates from DNA Making the replication fork I 2 opposite strands of ssDNA 0 leading uninterrupted 0 lagging interrupted I m directions leading strand one way is lagging the other I Fork structure dictates Okazaki fragment formation on lagging More replication fork DNA primese RNA orirr39iger DH r ligese DNA Polymerase iPolori Lagging 7 i A g m 4 p pr v 1 I m r q 371 j ililllllll 1 393 39 Leading 7 strand 4quot Topoiso merese 3r DNA Polymerase iPoIETJ Hellicese V Single strand 7 Binding proteins I ssDNA strands kept separated by SSBs single stranded DNA binding proteins 0 tetrameric binds ca 33 basestetramer 0 used in m DNA dependent processes I Recruitment of Topoisomerase II essential to relaX DNA tension 0 gyrase tetramer of 2 GyrA and 2 GyrB subunits 0 only in prokaryotes target of some antibiotics Gyrase induces negative supercoiling G SEgmer39rt VJ Eyre5e Binds G and T segments in DNA strands cuts G segment with power of ATP Passes T segment through opening DnaB recruits PolIII DNA polymerase Copyright The McGraaN Hilll Cormpan es lino PermissionI required for reproduction our display 3 clamp Core enzyme 088 DnaB he case Core enzyme polymerase is 3 subunits a the polymerase e proofreading editing function 3 to 5 exonuclease activity q helps e work t subunits tether core enzymes b clamp dimers tether core enzyme to DNA essential for processivity ca 15000 basesmin Multisubunit g complex Why a lagging strand clamp loader I When DNA PolIII hits downstream RNA primer it falls off DNA I Does NOT leave a compleX I a compleX must load new clamp to re associate DNA PolIII I Used clamp is discarded The sliding clamp I binds and slides freely on DNA I encircles DNA sticks directly to a subunit I confers processivity to PolIII I also important in DNA repair I conserved structure throughout biology Gamma complex Clamp loader Must hydrolyze ATP for activity Conserved throughout biology Problem with fragments Copyrlghm The Imu 39 nnc 39 requnneu for reuroduct otn or d splay 3 5 Parental strand 2 11gt 5 3 Lagging strand Okazaki fragmehts chk with RNA primer DNA polymerase II removes RiNA primer dHNTFZs and fills gap with iDNA rmi CK remains or NAD DNA Iizgase lliirwks Olkazakti fragme ts together by seallingi nick AMEF F Pi or lNlMlN 3 5 I small gaps between fragments nicks I RNA in DNA I fixed with DNA poll 0 5 to 3 exonuclease activity I Sealed with DNA ligase DNA Pol I I Major enzyme for nick translation I SLOW 10 20 nucsec O PolIII lOOO nucsec I Proofreads I Single subunit Also RNaseH I non essential RNase 5 to 3 exonuclease I only works on short RNA sequences I specifically binds DNARNA hybrids 0 no RNA or DNA binding alone 0 no activity on above I not the main player in Okazaki Fragment removal Termination min uch u l39lll39l EFLI I39I39Ill39i n m5 I lf rrri I I Termination sites are ter binding sites 0 10 sites IBind a single protein Tus in a directional manner I Anti helicase activity Tus has antihelicase activity I Knocks DnaB off of DNA I No unwinding no progression I Everything falls apart Tus activity is DIRECTIONAL 3 ILI SETEFE 31 Flquot pa ll Tug is 9 k MODEL of Tus activity I Permissive and non permissive faces I STALLED compleX waits for other side to join I Why do we need 2 directions Replication in both directions Finishing up Resolution of DN catalyzed by topoisomerase IV G segment 2 ATP 1 lt Core type II topoisomerase strand passage mechanism Upper clockwise from top Type II topoisomerase blue salmon and yellow binds gate segment DNA green and subsequently captures a transfer segment DNA pink to red denoting movement of the DNA followed by the binding of two ATP molecules which close the N terminal gate yellow This is followed by double strand cleavage of the gate strand and passage of the transfer strand across the cleaved DNA and through the enzyme The transfer strand and products of ATP hydrolysis are then released as the enzyme resets for another enzymatic cycle This core strand passage mechanism is coupled with substrate specificity to achieve different topological activities Gyrase Lower Left blue salmon and yellow with purple GyrA CTD wraps DNA around its GyrA CTD resulting in the formation of a left handed DNA crossing which is converted into a negative supercoil after passage of the transfer segment red through the gate segment green Topo IV Lower Right blue salmon and yellow with purple ParC CTD unlinks catenated DNA molecules and relaxes positive supercoils more efficiently than negative supercoils Lecture 17 Transcription Basics in Ch 12 Learning Objectives I Understand RNA polymerase subunits and activities Understand gene structure Understand the steps of transcription Learn different mechanisms of transcriptional termination Typical gene structure in DNA Copyright The McGraw lliil Companies Inc Permission required for reproduction or display RNA poiymerase recognition site RNA polymerase binding site Pribnow box Coding strand Template strand 35 i0 1 539 i i 339 DNA 3 i l I l 5 g I J I Jll I Jll 1 Ill l Promoter Leader Coding region Trailer Terminator Transcription Start Direction oi transcription gt a Shine Dalgarno G sequence or A AUG mRNA 5 39 I 39 3 Leader Trailer Translation start Translation initiation codon stop Gordon b I Promoter region defines RNA polymerase binding site important 10 35 seq I 1 nucleotide defines the start of mRNA synthesis I coding region encodes the protein information or not I untranslated regions have some information here OK We want to make some mRNA HA F39ol yme raise Eda m HTD I We need some RNA polymerase I Note multisubunit structure and DNA contacts I EMA Polyme raise ada m HTD 5 subunits in functional RNA polymerase b subunit involved in ribonucleotide chain elongation directs DNA w b b subunit has the active site for nucleotide chain polymerization a subunit forms a dimer 2 in active enzyme Major site of regulation can bind DNA 2 important domains the NTD N terminal domain and the CTD C term binds to core enzyme via b subunit s subunit directs promoter specificity directly contacts 10 and 35 sites in DNA w subunit not essential clamps RNAP together stability Getting to the promoter the factor EDIE ENE3955 gp ymeraa e Sigma EEiEtDE HHF39 pa lym ras fh remgym RNAP eXists as m enzyme in solution low affinity for non specific DNA Directed to promoter by binding sigma factor making holoenzyme increasing specificity Specificity lies in DNA binding sites so multiple sigma factors for each sequence s70 housekeeping s54 nitrogen starvation s38 stationary phase s32 heat shock s28 agellar regulation s24 extracytoplasmic stress s19 iron acquisition New sigma factors regulate regulons multiple co regulated genes More factor information I Does gtknotilt bind promoter DNA by itself 0 needs RNA polymerase holoenzyme I E 601139 has 7 but others have gt20 Usually part of the ECF extracytoplasmic function family I Can be regulated by anti sigma factors and other ways we ll talk about this eventually O transcriptional O sequestration 0 complete removal OK Got to the promoter Now what CCCCCCCC 4 Promoters have different af nities for RNAP kf RFD 0131 compiles I R RNAP I P promoter region I KB binding constant I kf isomerization constant Stronger promoters have bigger KB values and m kf values Opening the DNA heliX I RNAP has DNA binding channel where I Binds covers promoter from 70 to 20 I Conformational shift closes channel I Melts DNA ATP dependent Obviously RNA is not DNA I OH at 2 position of ribose is the difference I AT in DNA is replaced with AU in RNA uridine I All other basepairs are the same I Why U in RNA T is methylated U Promoter clearance I Most or many transcripts AB ORTIVE fail in first 10 nt I M clear initial promoter once past 21 nt no longer slips I Can you think why this is the case Think of RNAP affinities for DNA Elongating Open complex follows RNAP RNA separates from DNA follows a tunnel out of the enzyme DNA helix reforms on other side of enzyme Sigma factor is lostrecycled within 80 nucleotides NusA elongation factor binds RNAP where 3 left RNAP doesn t just go BEFORE SITE FAST CONFORMATIDN E55 CLOSED inquot SL 0 w IN TERMEDIA TE OPEN I n h i b its mil 36s pausing ua Ki 7 more back I T quotquot17quot I tracking 39 Stimulates pausing ARREST CLASS I PAUSE CLASS PAUSE TERM NA WON when halted p rho dependent termination I site in mRNA called rut site rho utilization O C rich 0 no 20 structure I Hexameric 6 protein Rho binds rm 0 ATPase 0 helicase Rho travels TOWARDS RNAP paused by NusA destabilizes RNA DNA hybrid helicase Rho loopsthreads RNA 0 NusA while elongation factor enhances pausing at step loop structures remember rho 0 outside of RNAP recently synthesized O Currently synthesized RNA MUST be U rich U A bond Why The termination stem loop 0 inverted repeats Another way to stop GC rich end of genes 2 structure usually precedes stretch of A or U in RNA disrupting stem formation disrupting termination also RNAP speed An experiment 11 regulation of cellulose degrading enzymes in Clostridium thermocellulansNataf Y et al PNAS 20101071864618651 Identify factors and genes to regulate 0 Sequence similarity to Bacillus factors 0 Genes for cellulose degradation known not important to know for you 0 Make templates of putative important promoters 7 proposed sigma factors for cellulose regulation Table 1 grantee pairs pmpmedl ta partieipate in regulating eellmlesamal genes efan ee gene39s Ptarmigan amtiee Citermiinall Target denim locus frag dumain aa rerli n uuegrii sensing damain palyeacchari es allege Eitheg 52 Gem Cellllullmer u sgllz Cihe 25amp 6 came Cellllullnae di egll Eithe m ea 5 em a dyad Fleetiin ame sgll l Eitheg l l 52 eema Cellllullnaer a iesgle Ethan area 50 came erabiimxylen aEesgre amalgam19 51 eel1a xylene eellllullnaer Elisang cane em1 an SHE Cellllulltaier Hat inrJuding the transmemhrane domain Cen rmed experimentally QT 31 antisigma factor Binds sigma factor prevents functional association with RNAP many are regulated in this manner The assay runoff transcription Promoter analysis of sigIl rsgIl and sigI6 rsgI6 operons The top panels show the mapping of the 539 ends of sigIl rsgIl A and sigI6 rsgI6 B transcripts determined by the 539 RACE technique Arrows indicate the transcriptional start site Framed letters are the suggested 35 and 10 sequences of the O factor binding site The bottom panels present the identified sigI rsgI and celS promoters asterisk as well as other putative promoter sequences of several cellulosome related genes Nucleotides similar to the sigI rsgI promoters are shown in bold Numbers indicate both the distance in nucleotides between the 35 and the 10 promoter regions and between the 10 promoter region and the start of the ORF GH glycoside hydrolase CE carbohydrate esterase CBM carbohydrate binding module Docl dockerin type I Coh2 cohesin type II SLH S layer homology module In Vitro transcription from the sigIl and celS promoters Promoter ceIS sigll rsgll RNAP Sigll ngllN M M M lt celS P4 494 I 301 301 244 sigll P 1 gt Spots are radioactive mRNA sizes correspond to predicted transcript length In Vitro transcription from the sigIl and celS promoters Runoff transcription reactions were performed using DNA fragments containing the sigIl or celS promoters and C thermocellum RNA polymerase RNAP preincubated in the presence or absence of 011 or 011 and ngIlN The asterisk indicates an in Vitro transcription reaction in which the amount of the celS promoter containing DNA added was increased from 0017 to 0068 pmol The size markers M are labeled single strand PCR products with the indicated nucleotide length Proposed mechanism for the activation of alternate 0 factors by extracellular polysaccharides OFF l NON Polysaccha ridles l gt l gt siglsig24C rsgIrSI3924C Celltulosomall get rites Proposed mechanism for the activation of alternate 0 factors by extracellular polysaccharides The ngIRsi24C transmembrane proteins red contain an extracellular carbohydrate active module CBM3 CBM42 PA14 GHlO or GHS and an intracellular anti O peptide domain In the OFF state the anti O domain interacts strongly with the alternative 0 factor blue thereby inactivating it In the ON state extracellular polysaccharides green interact with the CBM which in turn induces a conformational change on the intracellular anti O domain resulting in the release of the alternative 0 factor The 0 factor is now free to interact with RNA polymerase RNAP and promote transcription of the O dependent promoters Note that the 0 factor also promotes transcription of its own bicistronic operon which includes the cognate rsgIrsi24C gene Lecture 18 Gene Regulation Learning objectives 0 Re learn lac operon regulation add am and trp regulation to the list to know 0 Be able to understand regulation of a compleX multi part system if I give you all of the regulation conditions on a message can you tell me what the output is Proteinmediated transcriptional regulation 0 Activators O enhance transcription from promoters 0 most enhance affinity of RNAP for promoter region trap it 0 Repressors O um they repress transcription 0 tend to m binding sites at promoter for RNAP Basics of transcriptional control 0 Induction activation 0 Requires binding of an activator protein or removal of a repressor protein 0 Both types can be regulated by other ligands molecules proteins 0 Repression O Requires binding of a repressor protein or removal of an activator protein 0 see above Positive control activation Copyright the VcGrawHill Companies Inc Permission required for reproduction or display Indlucer Activator l ainding site No transcription or Activator protein c Positive control of an inducible gene Transcription occurs Activator protein RNA quot polymerase No transcription Inhibitor or Activator protein gtgt Transcription occms Activator protein C Positive control of a repressible gene Negative control repression Copyright the MCGraw Hill Companies Inc Permission required for reproduction or display RNA polymerase DNA P ro mote r Operator Transcription occurs Repressor No transcription or protein Inducer Reprressor protein a Negative controi of an inducible gene RNA polymerase gt Corepressor iNo transcription Transcription occurs or e Repressor protein aporepressor b Negative control of a repressible grerne Regulatory decision pathway Copyright the McGraw Hili Companies Inc Permission required for reproduction or display Repressor protein Regulatory decisions Biosynthetic enzymes End product or pathway present Catabolic enzymes Substrate of pathway present Yes Yes i Preferred carbon and energy source present No Yes Synthesize Do not enzymes synthesize Syntihesize enzymes enzymes Geneoperon Geneoperon Geneoperon on Off on Operons allow regulation of many genes Copyright The lMcGrawHiill Companies inc Permission required for reproduction or display Initiation codon Termination cordon usually AUG UAA UAGr UGA 5 36 Leader Coding region 1 Spacer Coding region 2 Trailer a iPolycistronic rnFiNA Initiation Termination codon codon 5 Leader Coding region Trailer b Mionocistronio rmRiNA 0 Single promoter at beginning of operon can co regulate related genes 0 Can be INTERNAL promoters in operons for additional regulation 0 Many times protein regulator for operon is encoded within operon or otherwise associated with it 0 guilt by association The Feared and loved lac operonone millionth time right Copyright the McGrawHill Companies lino Permission required for reproduction or display Regulatory gene lac operon I r quot E coli E is f gt7 39 i W7 J romosome Encodes lactose Encodes lac Encocies alactosidase 130 Diromo fer CAP Slte l Operator g g permease galactoside terminator lac promoter transacetylase O encodes enzymes required for the import and degradation of the disaccharide lactose 0 no lactose around Don t make the enzymes 0 First level of regulation negative control of operon transcription through LacI repressor LacI repressor hides RNAPbinding site in Lac promoter Copyright the McGraw I lill Companies Inc Permission required for reproduction or display Binding of lac repressor lac repressor tetramer lac repressor tetramer a Possible DNA loops caused by c the binding of the lac repressor 3 operator sites 01 is the main operator and must be bound by LacI to inhibit transcription DNA is bent or looped due to binding of LacI when additionally bound to 02 or 03 promoter hidden by 0103 loop Structure blocks RNAP progression 0102 bend LacI is a functional tetramer 4 The cells see lactose Copyright C the McGraW Hill Companies Inc Permission required for reproduction or display H Lactose Lactose per mease They make allolactose as a side reaction of bgalactosidase The Signal Allolactose is the inducer of lac Copyright 3 the MoGraw Hill Companies Inc Permission required for reproduction or display lac lac operon regu iatory r 39 1 gene Promoter Operator lac repressor binds to the operator and inhibits transcription lac repressor active 3 No lactose in the environment Copyright the McGiraw Hill Companies Inc Permission required for reproduction or display RNA polymerase Transcription Polyoistronio B gai actosidase Lactose Gaiactoside perm ease transacetyiase i V Ailoiactose The binding of aliolactose prevents 4 the lac repressor from binding to the operator site b Lactose present 0 Separate binding Site in LacI for allolactose not same as DNA binding 0 Binding induces conformational change in repressor no DNA binding lac is also positively regulated Regulatory gene Copyright tlhe McGraw Hilll Companies Inc Permission required for reproduction or dispiay lac o peron E coli r 1r ye r39 lac promoter CAP site Operator lac promoter OOO CAP site CAP senses general carbon source utilization state responds to CAMP levels in the cell 1 Wmmosome Encodes lactose Encodes lac permease galactoside terminator transacetylase Encodes B galactosi dase DNA binding site for the Qatabolite activator protein CAP 0 Main carbon source sensed by CAP is glucose Back to the PTS Copyright C the McGraw Hill Companies inc Permission required for reproduction or display El Pyruvate El 39HPr The high energy phosphate of PEP is transferred Via E1 to HER and from HPR to EIIA Phosphate is then transferred to incoming sugar via EHB Mannitol i P s L 5E gtr Mannitol HPr Giucose Ii if I 39 3quot Cytoplasmic E Periplasm matrix Copyright the MoGraw Hill Companies Inc Permission required for reproduction or display I When glucose is avaiiable the phosphate of PEP is transferred to EliA by way of Ell and l iPr EIIA then transfers the phosphate to EilB which in turn transfers it to the incoming giucose PEP EI XiIpf l illllliill iii ii i H IIIQICII l 3914 E H H gt g ql I is h i v ilrw i i iii quot l L l i Mquot u u Eurquotl 4 o i x i P yruvate EI l iiPr iEMA 043139 When glucose is not availabie 4 T 5 l the phosphate cannot be transferred guiis 39 1 to EllB and instead remains on EIIIA V k assquot iv EiAP activates adenyl cyclase I C in l AMP 1 f or and c is made ATP Q s F4 3952els CAMP O No glucose EIIA P transfers P to membrane bound adenyl cyclase AC 0 Makes CAMP from ATP low glucose high CAMP Can order sugars in this manner CAMP is required for CAPzDNA binding 0 important second messenger in prokaryotes eukaryotes 0 release of pyrophosphate PR 0 inversely proportional to glucose concentrations CAP bends DNA when bound Copyright Q the McGraw Hill Companies Inc Permission required for reproduction or display 5 3 Recognition helices a CAP binds inverted DNA repeats 3 T u an EFF AA A i 1 Invert Repeats A r I T I A I A V I A I T I Flecegnitien helices CAchAMP recruits RNAP A mh Dual signals compete at lac Copyright C the iiIcGraw Hill Companies Inc Permission required for reproduction or display Aliolactose D A q Transcription occurs W 39 CAMP Binding of RNA polymerase CAMP Transcription to promoter is enhanced is blocked R b CAP b re ressor 2332 y c Neither lactose nor glucose y p a Lactose but no glucose Allolactose Transcription 1 is inhibited by Transcription is inhibited lack of CAP Inactive by lack of CAP and presence of repressor Repressor d Glucose but no lactose inactive Inactive b Lactose and glucose Dual signals compete at lac no glulactose high cAMP from no glucose LacI still stuck to operators CAP binds cAMP recruits RNAP but OFF Glucose only low cAMP from glucose LacI still stuck to operators CAP cannot bind DNA to recruit RNAP Mutational analysis of the lac machinery O Mutations can be made that alter either protein or DNA function 0 Mutations in LacI and operator sequences are useful for understanding transcriptional regulation The important mutations O LacI null protein cannot bind operator DNA 0 LaclS super repressor protein cannot bind allolactose can t be induced 0 lacOC constitutive operator sequence cannot bind Lacl Lecture 19 Gene regulation Take 11 lac ara trp riboswitches Learning Objectives 0 Understand how lac is regulated in as or in trans applicable to many systems hint hint 0 Understand corepressors trp and bifunctional repressoractivators ara O Riboswitches Back to lac mutations O LacI null protein cannot bind operator DNA 0 LaclS super repressor protein cannot bind allolactose can t be induced 0 lacOC constitutive operator sequence cannot bind Lacl What do these mutations do What do these muta ons do lac11quot a the MEGTEWHIII ampanres lm mesm n requIred far rerlr39a mtrun m display FEE g u a tEry gene I39ali uperun 39 39 quotr 739 7 5633 mf39l39iCl Qr I E c da ngala tggi agE Encodes Language E ncudea 39 Arrraw u errrreaase gal Ct EidB tEErE39I I39HI39ISIeEH39 aran acaertyla e I c 39 I rca we 1 3 1quot Lacll mtuta u in DINA Sequence in Dp EF39EtDF can t bind represg r lac traunscripti n AELW39 f39S 3N1 if CAKE 31AMP available Molecular basis of lacOC mutation Molecular basis of acCDC m uta en wild tyne JaeIS seque ee Hormel seque ce E possi bale fan39ch muta e s Each SilthEquot mutation is SUi F EZEEVE IH te diSJrUHDt Lecl dlimezrftetrenmer binding die see the 2 SitE S ilher e What do these mutations do IMUE RTEE What do these mute ens Ia 1395 id true Hllll LEVEL Pear 39 39 rl39ar cur display FEEg39uEafbsry gel IE ran pem r r I I i for 77 553 E5 3 Lei H I 39 39 W V V r 7 r E E I65 qu m CAP Eng J Iguassth Er Icc z a s lygalaatusndase pgrmease g rul ctDSi a1 prIZIr l IDtEr La cl e I D a etese m r l Uta n er rlcls rrli 39 lac termunane EraJ IS E CaryI359 Fat traunscr39 p on In 39injducer bai39r39lcl i39r lg panCket in llLac cran it i39nduaced e aELWAVS UPI ever m with What do these mutations do Ia etc5e What do these muta ens do the MeGr wanI Games Ira Peimjsmn reqmre rl39ar FE r u CEn Dn 3quot display RE QUE a t w 95quot I39aI 7r 39 I M r V r 560 7 r a 55 r r s V V r gummomma Enumes lj galagtgai ase Enosdeg lactose Erica es 39 J permease Q l 39CtDSiQE a IDIEETZDS E termuna aer arans a39 Gertylase lac Summary m1utatier1 In DINA binding pocket 39in Lacl ca t be a represger Farr tremngcrmgptien AELWAVS DNi given enough C M P Z AP ferC Su mmarv meeeu ring Inc trenserip en e glluceee n g lueese glueese glimese 1 me lleeteee leetese me Heetese leetese lid turpe in erme Ii 7 r r In ECquot hu39ig h h igh wee k wee k neme ne e week In er high ig h 39wee week f CfS mene EH IIE FII39DF IE39 name How to tellll the difference between 51505 and fear Supply Leel pretein in trans Expressing genes in trans Expressing genes m trans Wk H mm eillezne l geese emite Steele zreplllmeeti mg eetree remeeemel DNA imeleew le eleemfnd eilll regunlleterv mznfetr metiem p ESEI t em a wwm mmn pleemidl we can treeseribe and enzyme er 10a Wi 39lr39W i tr a Ii li e llew fer eepreesiem ef gee e5 et a Ganammmmnm gleee5 etllher them the e srelmeeeme E gem irm ease ef a 1m Lmtea it lm vacquot 39 gang39Llnear wenter l m il mmmwmmmt 39 Inn new case empress Leel in siren539 in different fee mmtemt beieikgiremmdler the 7r Cheek ee tlfai f lptl l Ievellls eel Supplying Lacli or 060 in frees fune er ial lLaeli Example ledquot mutar it en bacterial ehr emespme ptherwise wildt39ype all ether genes pprmal what dupes this mea his is ON 39witheut laetpse Supply plasmid with nprrnal Laeil tp sell in trans Lael pa r ig arpur id frpr plasmid ear i new REPREEE ires without laetpse arid regulate nprmallyl Pirpiteiiin Lael is freely diffusible lir l cytpse l fact DNA is net in trans e eli ereint locations in Cis e same ileca mns same D Nf h molecule Supplying Laci or lacO in frail15 FU i lal lLasii Esamplei fast muta tl er bacterial shrpmpseme p tghemuri se wiletype all ether genes perm ian what dees this mean isms DIN wi theu t lastpse same as previpusii Supply plasmid with hermal Lasi ts sell is trsns Lasi pa r ig arpur id frem plasmid spurts sa net blips ias and is is still CINE Knew wihere tihe m uitaitiens are Can we tell the difference between MEGS and leaf UK New Suipili yquot lieCO in trans rigiht Supplying Lacl or fact m frat3915 1 Knew wlhere the mutations are ain where the structural genes are rue preteinl a a fact in trans EIEm IEE Fas r c mutaritj en bacterial Ehrerneseme etherwise wild type all ether genes ruermsl what dees this mesh fen is DH wi theut lactese 53 quotme as previeusll K Jainrt f lrget 1 nc Gina SupJew plasmid with hermal mica te cell in trans Lac if can s5 Eiin d Din plastm id Ias sits en plasmid seuree teensat rescue Fae en Ch remeserne arid slain is still DIN Lac regulation summary 0 lac has positive CAP and negative LacI regulatory components 0 allolactose is the LacI inducer knocks repressor off DNA cAMP is the CAP inducer allows CAP to bind DNA 0 LacI functions in trans only 6160 cannot am operon controlled by AraC protein 0 am structural genes araC araBAD encode an activatorrepressor and arabinose degradation enzymes respectively 0 AraC protein is like LacI but acts as an activator AND a repressor Acts by looping DNA 0 arabinose is the inducer molecule are regulation p Sl39tl UE a nd negatwe pmmwmwmwmw reC binds are epereter in 7 absentee e F arebi39rIese and lirllss tr e seEenid AraC prete ini beund tie 39F 39E epereter upstream IlHA le epliesl tra HISI r39i strive n of he FUEAD blacked Ilri prese ce EFF are bi39riese rst dimer ises tent are arid re i arid Certh interact lenig dista rice are bli39riese beurid Jarret dimer is CTIUATDR Fer ere n and armquot fi iyr nrifl u 1E r l tii l iril EI I r l i lil39 I l39 Eri39 l il lii39 lil Regula on with a eorepressorztrp Erwin3hr EH11 Wag4H EMW In Eilqlill39i39 il For reprndul m n5 lming Gemrqu Ema H f nE39IIII Ewemm Parmeem neamred Ear nE mdu m Iii lmy fry epemn r Attenuater 1 EHUEF ICE be r PM Vlyl nErEISE g Tr etnphan I r Mamiee 7 7 V 39 ful l FE39FZIFE39SEQF39 Cemereeeerreereeeer Eel Lew lr39y t pi39i n entersi tif i39iGEi i lii i39l l the em re I39m Kepieren mum quotmm 339313913959 3951mm EGFEWEEWF TEDFE ESQT tiffquot 1 3 weereter rquot Tira Stsripimr l 39 tie33 menacinghem USUan rE prESSD F its High tW EQ h l IEUEISE 1MEF E3953I lecture limitiell me dle inactive F There is elmether lever Eff regimenem llhere the trpi e emmaten This is ier When twpt pha quot5 Evannab lequot It after we dhuseuee translated binds te TlFFJH 35 a eerepreeeer marking T r39pH active fer repreeeieri am and trp summary 0 Both operons regulated by another molecule arabinose tryptophan binding to a DNA binding regulatory protein 0 AraC activator AND repressor O TrpR requires eorepressor for repressor function Riboswitches O Depend on RNA secondary structure in mRNA for regulation 0 This mRNA structure has the ability to bind effector molecules that drive regulation 0 Usually in non coding regions of the mRNA leaders Example 1 rib operon in Bacillus Gene empressizen pin Gerla asipra39asnpri a li a 9 3 guitaristin FMH Menusum f i ll 77 LJuuu 3 I WEE 539 Trausnripiipn steps rib ape ran tra nscriiptien resiperids te flauiu WG Dl LliCIEDtidE FMML a iprpduet pf ribd auin During transeriiptieri er the 5 leader seuueriees upstream at rial3 mFtN t pan assume a particular stem illeeipw structure letth 5 pntaneeus feldliug Liner nip FIMIFIII eanditiansr an antitermiriater steim illeep is termed Example 1 rib operpn in Bacillus Gene empiressiren pin Gene empruasnd d fi Tit 3 RibsPlump FMHiMptibpluEsi gt 1 4r is fPr L vs 739 quot 1 r 2 V n p MM 7 u Anutermmateir i i T V am w r J V u 77 UUUU quot Transcrip tien h cemtinues Transcription steps lri preseriee at FMINI deri t need tie malte arwmere rib genele FMM llainds tan a 1 laps in the llleadler sedueripei Einding FMIN disrupts tine autitermiiuater stem iller ferimatieu ipauripalefilalueL aillliewing a terminater stem leap te fe rim Terminater steim illeep stretelh at Ll inn leader E feu knew Example 2 gly biosynthesis in Bacillus 0 Example of a T box riboswitch 0 respond to uncharged tRNAs 0 many aa biosynthetic operons are regulated as such in Gram positives O Uncharged tRNA binds to specifier loop prevents terminator stem loop a Ems Stem I003 structures in Iy 5quot UTR I G u an Iii Ll Esq a new 23 Speci er lamp has 1 CDntairns H Hal c agn Mfar regLulla rtziripr Emma ECId 1 7 r n N3 hm a I Terminaltar Hi this cssE GEE 32 aquot u i I a quotEllh 7 it g r r 7 EGG 5 ELE Structures farmsEll Enulld be IJ g V quotmaid g 53 WWW 39 fl f gig Anhtermmstar Ilaft DR 25 terminatnr right but Inth both a g magi How dices this wnrk and note 7 33 the stretch of Us after thE v39 emu V 33 tErmlnatnr lumpquot 3 quotill up u 391 I 39939 U a iLl iii u Qi 39 E In 339 u same sequence but A Gum dl erent structures Q FHE k ir39l and Grundy EDIE E Enid S r i g H rb Syn11 t EEC 112231 23 tRNA charging review 0 Amino acids activated via adenylation attached to tRNA 0 aminoacyl tRNA synthetases O Anticodon pairs with mRNA bases to add amino acids to growing peptide chain Back to gf y regula 39on T box 113 ll l Antlitermirlxator iarmirlatulr 7 R323 lth Ecrugun FeEm i nae13 r 1 3t i l i rilli Stem llll Hliagl n RMH charming ssharming OENLVV forums Ioo p termi at ioh Ioo p by bir idir ng N10 trangcrip Oh I rbO at empt39y39 tiRlNA acceptor er id TbOX riboswitch summary 0 Common method for regulation of amino acid biosynthetic operons in Gram positive organisms O Depends on uncharged tRNA molecules binding RNA 0 amino acid starvation Make More 0 2 different mRNA stem loop structures are formed an antiterminator and a terminator Think about it 0 What would happen if you mutated some of the antiterminator nucleotides in the riboswitch antiterminator stem loop preventing structure 0 Some of these riboswitches are CATALYTIC they cleave themselves What do you think the effector molecule does in this case Lecture 20 Learning Objectives 0 Steps in initiation of translation 0 Steps in elongation of translation 0 EF Tu cycle 0 Recycling of ribosome termination Translation 0 Synthesis of polypeptide from genetic information in mRNA ribosomes amino acids RNA 0 Regulated at many steps like transcription 0 Coupled translation and transcription Cotranslation Copyright The McGraw Hi ll Companies Inc Permission required for repro ducticn or disyp ay 0 Because of ribosome size minimal spacing is 35 nt Reading the Genetic Code 0 The ribosome 0 Binds RNA tRNA and directs peptide bond formation 0 Multi subunit contains RNA bound ribosomal RNA rRNA O 30S small 0 50S large 0 70S together The Ribosome components 0 30S from Thermus thermophilus 0 Protein in blue 0 22 subunits labeled Sl S22 0 only 21 proteins one is modified 0 RNA in orange 0 16S RNA critical for initiation 1540 nt Large subunit 0 50S subunit from Haloarcula marismortui archaeal 0 Protein in blue 0 34 proteins Ll L36 0 RNA in yellow SS 120 nt and orange 23S 2900nt 0 Catalytic site green Catalytic site of SOS subunit 0 A site incoming tRNA site acceptor polypeptide built at this site 0 P site A site tRNA containg nacent polypeptide moves to this site peptidyl 0 E site Exit site empty tRNA is discharged 0 Catalysis provided by 23S RNA Peptidw 508 subunit Arming tramsfer ase 7 7 r 39 39 39 39 r r r 7 antic centre 39 Decadii mg I rag ii on Ft MA EDS subunlit RNA in ribosome 16 0 Major structural role scaffold for 303 proteins 0 binds protein 0 interacts with large subunit rRNA 23S RNA 0 CONSERVED across prokaryotic species major role in phylogenetic studies 0 3 end contains the antiShineDalgarno sequence ShineDalgarno sequence iIDNH G 3 and of El 2amp2 2155 rHHA A quotL u 2 LI E PI27 Ll II 1 5quot 2222222 s i GAL u or U EGG AGE u u 3 accumulate A223 2 ugu u Mm UMeasamger FE39NA I A I V i i l a 4 H J Iete rg e la F39he geu P39 tlypeptude 3n 0 AGrich region 8nt upstream of start codon for gene AUG 0 Note not all organisms require SD sites AUrich regions can be recognized by the SI ribosomal subunit protein RNA in ribosome 23S and SS 0 In large SOS subunit 0 238 provides structural AND catalytic role 0 catalyzes transpeptidation reaction for peptide synthesis 0 SS stabilizes SOS unit still not well understood The Genetic Code Table 122 5311 Second Position U C A G UUU UCU UAU UGU U U Phe Tyr Cy UUC UCC Ser UAC UGC c UUA UCA UAA UGA STOP A Le 1131 UUG UCG UAG UGG Trp G UU CCU AU GU U His 3 CUC CCC CAC CGC C A Q C Leu Pro Arg g LE CUA CCA CAA CGA A 5 in Gin 3 CUG CCG CAG CGG G E 5 3 2 AUU ACU AAU AGU U a 33 A51 Scr E 3 AUG Ilc ACC 7 AAC AGC C 393 A A 39lhr quotlg L AUA ACA AAA AGA A Lye Arg AUG Met ACG AAG AGG G GUU GCU GAU GGU U Asp GUC 2I AI L L C G Val Ala Cly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G code the RNA form Codons run in the 5 to 339 direction Code is degenerate 0 Multiple codons for a single amino acid 0 tend to differ in 3 position wobble 0 Fewer tRNAs than codons 0 E 601139 has 56 tRNAs for 61 sense codons encode aas 0 Wobble helps reduce impact of mutations base changes in 3 position Copyright The McGrew Hill Companies Inc Permission required for reproduction or display I 5 lily O O C 3 C 3 lt ooe coo mRNA5 GGU 3 i GGC a Base pairing of one glycine tRNA with two codons clue to wobble Glycine nriFlNA codeine GGLJ GGC GGA GGG 5quot g 3 Glycine tRNA antioodons COG CCU COG 3 h 5quot b Glycine oodons and anticodons tRNAs are charged activated 0 Charged by aminoacyl tRNA synthetases termed aaRS 0 Recognize amino acid AND codon on tRNA 0 Contain proofreading functions enymatic mechanisms conserved across biology tRNA proofreading by a separate domain on aaRS EJEiaiiS h i i igli lil li El Chili5 ll iil iFEEeIlTETirFiEaJ 51 n Iih39iE Ii39l 4 a in adliEnyla Diizm E 2 classes Class I monomer Class II dimer 3 domains anticodon enzymatic editing The initiation cycle Free 308 has IF 3 protein bound to it prevents SOS binding IF 20GTP binds only N formylmethionine tRNA tRNAfMet delivers to 30S Initiation methionine Blocks N terminus see book 1238 30S IF2GTP2tRNAfM and IF l all bind mRNA around SD site IF l displaces IF 3 50S subunit clamps down hydrolysis of GTP loss of EF l and EF 2 tRNAfMet starts in P site Copyright The McGraw Hill Companies Inc Permission required for reproduction or display lF 3 until the appropriate time fMet Wet 30 subunit IF2 I 153 rRNA aquot i Q GTP k complementary region F mRNA binds the SOS subunit 39 39 39 Initiator tRNA lF2 binds GTP and fMettRNA and guides them to the P sit F1 of the SOS subunit lF l binds the SOS subunit causing lF3 to leave This creates the SOS initiation complex which can now bind the 508 subunit As the 508 subunit binds to the 308 subunit GTP is hydrolyzed lF 1 and lF 2 are released and the 708 initiatio 39 PI ncomple and GDP 3 l x IS formed Elongation Elongation factor EF Tu binds GTP charged tRNA but never tRNAfMet EF Tu2tRNAAA is delivered to the A site in the 50S subunit GTP hydrolysis on EF Tu must hydrolyze to leave Transpeptidation occurs nacent polypeptide on A site tRNA P site tRNA blank Elongation factor EF G0GTP interacts with ribosome blocks A site hydrolyzes GTP physically moves ribosome down one codon A site to P site P site to E site eXit Copyright The McGraw Hill Companies Inc Permission required for reproduction or display EFTu binds GTP and an aminoacyl RNA and brings the amino acid to the A Site of the ribosome P J GTP IS hydrolyzed as it does so v f EFsru Hub GTP l mRNA J Binding AAlFlNA lo A sile AArtRNArGTPrEFrTu complex EFiTs EFrTu EpTu s EFG GTP complex 39 m Translocallon involves Elie which binds GTP Hydrolysis of GTP provides the energy e ded m ove en osome a he x codon in the mRNA Transpeptidation 0 Nucleophilic attack by amino group from A site amino acid on carboxyl group of P site Chain entire peptide transferred to A site Copyright The McGrawHill Companies Inc Permission required for reproduction or display P site A site NHQ 39 39 o NHX l Nthk 390 P o oP o leN KN Ngt cl A N I N HO O OH oSi lt3 39 co Fin i H HQN C H T R z i0 R CH NH PSIte l ASIte HO OH O OH I i0 n 2 H NH I i0 Rn1 H TH i0 Fin EH NH EFTu recycling After tRNAAA delivery EF Tu is GDP bound EF Ts required to remove GDP from EF Tu Fresh GTP can bind EF Tu and displace EF Ts Newly GTP bound EF Tu can now bind next tRNAAA for 708 delivery Copyright The McGrawHill Companies Inc Permission required for reproduction or display EFTu binds GTP n a Inoac RNA and brings t I A t Hh u n 3 P R a AArtRNArGTPrEFrTu complex EFTS GTP a EF TU EF Tu GDP ZGDP EFrTs 9 EF TS 3 I Peptide bond formation mRNA we lt3quot 8139 39I ii 4 l t 1 A 1quot Termination Ribosome incorporates amino acids until we see STOP codons UAA ochre most common UAG amber UGA opal No tRNAs for stop codons but RF l UAA UAG and RF 2 UAA UGA proteins recognize stop codons RF 3 helps binding RF l or 2 binding makes the nucleophilic attack agent water peptide release from tRNA RRF not pictured and GTP hydrolysis required for dissociation of ribosome Copyright The McGraw Hill Companies Inc Permission required for reproduction or display AA tRNA Peptide chain g UAA mRNA 3DS IF73 Termination proteins mimic tRNAs Copyright The McGraw Hill Companies Inc Permission required for reproduction or display TwC stem Acceptor stem TWO loop D loop 339 acceptor end Variable loop Anticodon Stem


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