1Chapter 12Genes: Expressionand Regulation2DNA Transcription or RNA Synthesis• produces three types of RNA– tRNA• carries amino acids during protein synthesis–rRNA• component of ribosomes–mRNA• directs protein synthesis3Transcription in Procaryotes• polygenic mRNA – contains directions for > 1 polypeptidesFigure 12.14Transcription in procaryotes…• catalyzed by a single RNA polymerase– large multi-subunit enzyme5Transcription in procaryotes…• E. coli RNA polymerase– core enzyme = α2ββ′– holoenzyme = core enzyme + sigma factor (directs core enzyme to promoter)• Thermus aquaticus RNA polymerase– core enzyme = α2ββ′ω– holoenzyme = core enzyme + sigma factor6Thermus aquaticus RNA polymerase7Figure 12.25′→3Pribnow boxand –35regions are importantrecognition sites89synthesis continuesuntil terminatorreachedFigure 12.210Procaryotic terminators• two types– hairpin + 6 uridines– rho factor-dependent• lack polyU and often lack hairpinFigure 12.411Transcription in Eucaryotes• monogenic mRNA12Yeast RNA polymerase II13Transcription in eucaryotes…• promoters contain three common elements– TATA box ~ 30 bases before transcription start– CAAT box ~ 75 bases before transcription start– GC box ~ 90 bases before transcription start14heterogeneous nuclearRNAexon intronRNA splicingFigure12.5amonogenic15Figure 12.5bsplicing occurs in largecomplex called a spliceosome –contains small nuclear RNAscomplexed with proteinsRNA splicing165′ cap of eucaryotic mRNAFigure 12.617Ribozymes• RNA molecules with catalytic activity• e.g., self-splicing rRNA moleculesBox 12.118Protein Synthesis• translation– synthesis of polypeptide directed by sequence of nucleotides in mRNA• direction of synthesis N terminal → C-terminal• ribosome– site of translation– polyribosome – complex of mRNA with several ribosomes19Transfer RNA and Amino Acid Activation• attachment of amino acid to tRNA• catalyzed by aminoacyl-tRNA synthetases– at least 20• each specific for single amino acid and for all the tRNAs to which each may be properly attached (cognate tRNAs)20Figure 12.8complementary tocodon in mRNAsite where aminoacid is attached21Figure 12.922Aminoacyl-tRNA↓ rest of tRNAamino acidFigure 12.1123The Ribosome• procaryotes– 70S ribosomes = 30S + 50S subunits• eucaryotes– 80S ribosomes = 40S + 60S subunits– mitochondrial and chloroplast ribosomes resemble procaryotic ribosomes24The 70S ribosomehelps alignribosomewith mRNAother rRNAs –may havecatalytic role20 nmFigure 12.1225E. coli ribosomeFigure 12.1326Initiation of Protein Synthesis• involves ribosome subunits and numerous additional molecules– initiator tRNA– initiation factors (IFs)N-formylmethionine-tRNA – bacterialinitiator tRNAFigure 12.14archaea and eucaryotes usemethionine-tRNA27initiationcodon• the goal –position ribosomeproperly at 5′ endof mRNAFigure 12.1528Elongation of the Polypeptide Chain• elongation cycle– sequential addition of amino acids to growing polypeptide– consists of three phases• aminoacyl-tRNA binding• transpeptidation reaction• translocation– involves several elongation factors (EFs)29tRNA binding sites of ribosome• peptidyl (donor; P) site– binds initiator tRNA or tRNA attached to growing polypeptide (peptidyl-tRNA)• aminoacyl (acceptor; A) site– binds incoming aminoacyl-tRNA• exit (E) site– briefly binds empty tRNA before it leaves ribosome30Figure 12.16transpeptidation reaction31Transpeptidation reactionFigure 12.16catalyzed by peptidyl transferase32Figure 12.16translocation –threesimultaneousevents1. peptidyl-tRNAmoves from Asite to P site2. ribosomemoves downone codon3. empty tRNAleaves P site33Termination of Protein Synthesis• takes place at any one of three codons– nonsense (stop) codons – UAA, UAG, and UGA• release factors (RFs)– aid in recognition of stop codons– 3 RFs function in procaryotes– only 1 RF active in eucaryotes34Figure 12.1835Protein Folding and Molecular Chaperones• molecular chaperones– proteins that aid the folding of nascent polypeptides– protect cells from thermal damage• e.g., heat-shock proteins– aid in transport of proteins across membranes36Figure 12.19In bacteria…37Protein folding – eucaryotes versus procaryotes• domains– structurally independent regions of polypeptide– separated from each other by less structured portions of polypeptide• in eucaryotes– domains fold independently right after being synthesized– molecular chaperones not as important• in procaryotes– polypeptide does not fold until after synthesis of entire polypeptide– molecular chaperones play important role38Protein Splicing• removal of part of polypeptide before folding• inteins –removed portion• exteins –portions that remain in proteinFigure 12.2139Regulation of mRNA Synthesis• regulation of gene expression• conserves energy and raw materials• maintains balance between the amounts of various cell proteins• allows organism to adapt to long-term environmental change40Induction and Repression• inducible enzyme– level increases in presence of inducer• small molecule, usually substrate of catabolic pathway in which enzyme functions• repressible enzyme– level decreases in presence of corepressor• usually end product of biosynthetic pathway in which the enzyme functions41Negative Control• presence of regulatory protein (repressor) at regulatory site (operator) decreases mRNA synthesis• repressor proteins– exist in active and inactive forms– inducers and corepressors alter activity of repressor42an operonpromoteroperatorFigure12.23negative control ofcatabolic pathway• the goal –makeenzymes ofpathwayonly whensubstrate ofpathway isavailableusually substrate ofpathwaystructural gene =gene coding for polypeptide43negative control of abiosynthetic pathway• the goal –only makeenzymes ofpathway whenend product ofpathway is notavailableusually end productof pathwayFigure 12.2444Positive Control• presence of a regulatory protein (activator protein) at a regulatory region promotes transcription• e.g., lactose operon– regulated by catabolite activator protein (CAP) and cyclic AMP (cAMP)• CAP also called cyclic AMP receptor protein (CRP)45Figure 12.22β-galactosidase reaction46cAMP• binds to and activates CAPFigure 12.2747CAPFigure 12.29recognizeand bindregulatoryregion oflactoseoperon48Figure12.28levels vary