Genetics: From genes to genomes - Chapter 8: What genes are and what they do

*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or displayPowerPoint to accompanyGenetics: From Genes to GenomesFourth EditionLeland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. SilverPrepared by Mary A. BedellUniversity of Georgia*CHAPTERWhat Genes Are and What They DoCHAPTERPART IICHAPTER OUTLINECopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Gene Expression: The Flow of Information from DNA to RNA to Protein8.1 The Genetic Code8.2 Transcription: From DNA to RNA8.3 Translation: From mRNA to Protein8.4 Differences in Gene Expression Between Prokaryotes and Eukaryotes8.5 A Comprehensive Example: Computerized Analysis of Gene Expression in C. elegans8.6 The Effect of Mutations on Gene Expression and Gene FunctionFour general themes for gene expressionPairing of complementary bases is the key to the transfer of information from DNA to RNA and from RNA to proteinPolarities of DNA, RNA, and polypeptides help guide the mechanisms of gene expressionGene expression requires input of energy and participation of specific proteins and macromolecular assembliesMutations that change genetic information or obstruct the flow of its expression can have dramatic effects on phenotypeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Gene expression: the flow of genetic information from DNA via RNA to proteinRNA polymerase transcribes DNA to produce an RNA transcriptRibosomes translate the mRNA sequence to synthesize a polypeptideTranslation follows the "genetic code"*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.2Triplet codons of nucleotides represent individual amino acids61 codons represent the 20 amino acids3 codons signify stop*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.3A gene's nucleotide sequence is colinear with the amino acid sequence of the encoded polypeptideCharles Yanofsky – deduced key features of relationship between nucleotides and amino acidsGenerated large number of trp− auxotrophic mutants in E. coliDetailed analysis of mutations in trpA geneTrpA encodes a subunit of tryptophan synthetaseFine structure genetic map of trpA gene based on intragenic recombinationDetermined amino acid sequences of mutant tryptophan synthetase Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Mutations in a gene are colinear with the sequence of amino acids in the encoded polypeptideDifferent point mutations may affect the same amino acidCodons must contain >1 nucleotideEach point mutation affects only one amino acidEach nucleotide is part of only one codonCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.4aEvidence that codons must contain two or more base pairs Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Intragenic recombinationWild-type allele can be produced by crossing two mutant strains with different amino acids at the same positionFig. 8.4bStudies of frameshift mutations showed that codons consist of three nucleotides*Fig. 8.5F. Crick and S. Brenner (1955)Proflavin-induced mutations in bacteriophage T4 rIIB geneIntercalates into DNACauses insertions and deletions2nd treatment with proflavin can create a 2nd mutation that restores wild-type function (revertant)Intragenic suppression)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Different sets of T4 rIIB mutations generate either a mutant or a normal phenotypeCodons must be read in order from a fixed starting pointStarting point establishes a reading frameIntragenic supression occurs only when wild-type reading frame is restored *Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.5dCodons consist of three nucleotides read in a defined reading frame*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.6Counterbalancing of mutations (e.g. +1 and -1) can restore the reading frameIntragenic suppression occurs when mutations involve multiples of threeCodons consist of three nucleotides read in a defined reading frame (cont)Frameshift mutations alter the reading frame of codons after the point of insertion or deletion*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.6Cracking the code: Which codons represent which amino acids?Several technological breakthroughs in 1950s and 1960sDiscovery of mRNAIn vitro translation of synthetic mRNAsPreparation of cellular extracts that allowed translation in a test tubeDeveloped techniques to synthesize artificial RNAs with known nucleotide sequenceAllowed synthesis of simple polypeptidesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Cracking the code: Discovery of mRNA1950s, studies in eukaryotic cellsEvidence that protein synthesis takes place in cytoplasm Deduced from radioactive tagging of amino acidsImplies that there must be a molecular intermediate between genes in the nucleus and protein synthesis in the cytoplasmDiscovery of messenger RNAs (mRNAs), molecules for transporting genetic informationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Using synthetic mRNAs and in vitro translation to crack the genetic code1961 – Marshall Nirenberg and Heinrich Mathaei Added artificial mRNAs to cell-free translation systemsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.7aSimple polypeptides are encoded by simple polynucleotidesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.7bSynthetic mRNAPolypeptides synthesizedThe coding possibilities of synthetic mRNAsCracking the genetic code with mini-mRNAsNirenberg and Leder (1965)Resolved ambiguities in genetic code In vitro translation with trinucleotide synthetic mRNAs and tRNAs charged with a radioactive amino acidCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.8Correlation of polarities in DNA, mRNA, and polypeptideTemplate strand of DNA is complementary to mRNA and to the RNA-like strand of DNA5’-to-3’ in the mRNA corresponds to N-to-C-terminus in the polypeptide Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.9Summary of the genetic codeGenetic code has triplet codonsCodons are nonoverlappingThree nonsense codons don’t encode an amino acid; UAA (ocher), UAG (amber) and UGA (opal)Genetic code is degenerateReading frame is established from a fixed starting point – codon for translation initiation is AUGmRNAs and polypeptides have corresponding polaritiesMutations can be created in three ways; frameshift, missense, and nonsenseCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Experimental verification of the genetic codeMissense mutations are single nucleotide substitutions and conform to the codeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.10aYanofsky: Single-base substitutions can explain the altered amino acids in trp− and trp+ revertantsAmino acid in mutant polypeptideAmino acid in wild-type polypeptidePosition in polypeptideExperimental verification of the genetic code (cont)Yanofsky: Amino acid alterations that explain intragenic suppression of proflavin-induced frame-shift mutationsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.10bGenetic code is almost, but not quite, universal Virtually all cells alive now use the same basic genetic codeIn vitro translational systems from one organism can use mRNA from another organism to generate proteinComparisons of DNA and protein sequence reveal perfect correspondence between codons and amino acids among all organismsGenetic code must have evolved early in history of lifeExceptional genetic codes found in ciliates and mitochondriaCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Transcription: From DNA to RNARNA polymerase catalyzes transcriptionPromoters are DNA sequences that provide the signal to RNA polymerase for starting transcriptionRNA polymerase adds nucleotides in 5’-to-3’ directionFormation of phosphodiester bonds using ribonucleotide triphosphates (ATP, CTP, GTP, and UTP)Hydrolysis of bonds in NTPs provides energy for transcription Terminators are RNA sequences that provide the signal to RNA polymerase for stopping transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Transcription in bacterial cellsInitiation: The beginning of transcriptionRNA polymerase binds to promoter sequence located near beginning of geneSigma (s) factor binds to RNA polymerase ( holoenzyme) Region of DNA is unwound to form open promoter complexPhosphodiester bonds formed between first two nucleotidesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.11aTranscription in bacterial cellsElongation: An RNA copy of the gene factor separates from RNA polymerase ( core enzyme)Core RNA polymerase loses affinity for promoter, moves in 3’-to-5’ direction on template strandWithin transcription bubble, NTPs added to 3’ end of nascent mRNA*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.11bTranscription in bacterial cells Termination: The end of transcriptionTerminators are RNA sequences that signal the end of transcriptionTwo kinds of terminators in bacteria: extrinsic (require rho factor) and intrinsic (don’t require additional factors) Usually form hairpin loops (intramolecular H-bonding)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.11cThe product of transcription is a single-stranded primary transcript*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.11dThe promoters of 10 different bacterial genesMost promoters are upstream to the transcription start pointRNA polymerase makes strong contacts at -10 and -35*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.12Strong E. coli promotersStructure of the methylated cap at the 5' end of eukaryotic mRNAsCapping enzyme adds a "backward" G to the 1st nucleotide of a primary transcript*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Transcribed basesFig. 8.13Methylated cap – not transcribedTriphosphate bridgeProcessing adds a tail to the 3' end of eukaryotic mRNAs*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.14RNA splicing removes intronsExons – sequences found in a gene’s DNA and mature mRNA (expressed regions)Introns – sequences found in DNA but not in mRNA (intervening regions)Some eukaryotic genes have many intronsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*The human dystrophin gene: An extreme example of RNA splicingSplicing removes introns from a primary transcript*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.15RNA processing splices out introns and joins adjacent exonsShort sequences in the primary transcript dictate where splicing occurs*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.16aRNA processing splices out introns and joins adjacent exons (cont)Two sequential cuts remove an intron*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.16bSplicing is catalyzed by the spliceosome*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.17Alternative splicing can produce two different mRNAs from the same gene*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.18aTrans-splicing combines exons from different genesOccurs in C. elegans and a few other organisms*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.18bTranslation: From mRNA to proteinTransfer RNAs (tRNAs) mediate translation of mRNA codons to amino acidsTranslation takes place on ribosomes that coordinate movement of tRNAs carrying specific amino acidstRNAs are short single-stranded RNAs of 74 – 95 ntEach tRNA has an anticodon that is complementary to an mRNA codonA specific tRNA is covalently coupled to a specific amino acid (charged tRNA)Base pairing between an mRNA codon and an anticodon of a charged tRNA directs amino acid incorporation into a growing polypeptideCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8**Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.19aSome tRNAs contain modified basesThree levels of tRNA structureNucleotide sequence is the primary structureSecondary structure (cloverleaf shape) is formed because of short complementary sequences within the tRNATertiary structure (L shape) is formed by 3-dimensional foldingCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.19bAminoacyl-tRNA synthetases catalyze attachment of amino acids to specific tRNAsEach aminoacyl-tRNA synthetase recognizes a specific amino acid and the structural features of its corresponding tRNA Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.20Base pairing between an mRNA codon and a tRNA anticodon determines which amino acid is added to a growing polypeptide*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.21Wobble: Some tRNAs recognize more than one codon for the amino acid they carry*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.22A ribosome has two subunits composed of RNA and protein*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.23aMechanism of translationInitiation stage - start codon is AUG at 5’ end of mRNAIn bacteria, initiator tRNA has formylated methionine (fMet) Elongation stage - amino acids are added to growing polypeptideRibosomes move in 5’-to-3’ direction along mRNA2-15 amino acids added to C terminus per secondTermination stage - polypeptide synthesis stops at the 3' end of the reading frame Recognition of nonsense codonsPolypeptide synthesis halted by release factorsRelease of ribosomes, polypeptide, and mRNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Different parts of a ribosome have different functionsSmall subunit binds to mRNALarge subunit has peptidyl transferase activityThree distinct tRNA binding areas – E, P, and A sites*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.23bTranslation of mRNAs on ribosomes: Initiation phase in prokaryotesRibosome binding site consists of a Shine-Dalgarno sequence and an AUGThree sequential steps: small ribosomal subunit binds first, fMet-tRNA positioned in P site, large subunit bindsInitiation factors (not shown) play a transient role *Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.25Translation of mRNAs on ribosomes: Initiation phase in eukaryotesSmall ribosomal subunit binds to 5' cap, then scans the mRNA for the first AUG codonInitiator tRNA carries Met (not fMet)*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.25Translation of mRNAs on ribosomes: Elongation phase*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.25Addition of amino acids to C-terminus of polypeptideCharged tRNAs ushered into A site by elongation factors (not shown)Polyribosomes consist of several ribosomes translating the same mRNASimultaneous synthesis of many copies of a polypeptide from a single mRNA*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.25Translation of mRNAs on ribosomes: Termination phaseNo normal tRNAs carry anticodons for the stop codonsRelease factors bind to the stop codonsRelease of ribosomal subunits, mRNA, and polypeptide*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.25Posttranslational processing can modify polypeptide structure*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.26(a) Cleavage may remove an amino acid(b) Cleavage may split a polyprotein(c) Chemical constituent addition may modify a proteinDifferences between prokaryotes and eukaryotes in gene expressionOverview*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Table 8.1Differences between prokaryotes and eukaryotes in gene expression (cont)Transcription*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Table 8.1 (cont)Differences between prokaryotes and eukaryotes in gene expression (cont)Translation*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Table 8.1 (cont)A computerized analysis of gene expression in C. elegans: A comprehensive exampleC. elegans is an ~ 1 mm roundworm that lives in soil Ideal organism for genetic analysisSmall size, short life cycle, prolific reproductionC. elegans genome contains roughly 20,000 genes15% encode components of gene expression machinery60 genes encode parts of ribosomes300 genes encode transcription factors695 tRNA genes, 100 rRNA genes, 72 snRNA genesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Landmarks in the collagen gene of C. elegansNucleotide sequences of genomic DNA and all mRNA can be obtained for many genes (described in Chapters 9 and 10)Allows detailed analysis of gene structure based on computational analysis of sequencesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.27aSequence of a C. elegans collagen gene, mRNA, and polypeptide*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.27bTypes of mutations in the coding sequence of genes*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.28aMutations in the coding sequence of a gene can alter the gene productMissense mutations replace one amino acid with anotherConservative – chemical properties of mutant amino acid are similar to the original amino acide.g. aspartic acid [(-)charged]  glutamic acid [(-)charged]Nonconservative – chemical properties of mutant amino acid are different from original amino acide.g. aspartic acid [(-)charged]  alanine (uncharged)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Mutations in the coding sequence of a gene can alter the gene product (cont)Nonsense mutations change codon that encodes an amino acid to a stop codon (UGA, UAG, or UAA) Frameshift mutations result from insertion or deletion of nucleotides with the coding region No frameshift if multiples of three are inserted or deletedSilent mutations do not alter the amino acid sequenceDegenerate genetic code – most amino acids have >1 codonCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Mutations outside the coding sequence can disrupt gene expression*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig. 8.28bLoss-of-function mutations result in reduced or abolished protein activityLoss-of-function mutations are usually recessiveNull (amorphic) mutations – completely block function of a gene product (e.g. deletion of an entire gene)Hypomorphic mutations – gene product has weak, but detectable, activityCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.29Loss-of-function mutations result in reduced or abolished protein activity (cont)*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Some loss-of-function mutations can be dominantIncomplete dominance – phenotype varies with the amount of functional gene product (Fig 8.30)Haploinsufficiency – phenotype is sensitive to gene dosage (i.e. 50% of gene product) (Fig 8.31a)Fig. 8.30Fig. 8.31aLoss-of-function mutations result in reduced or abolished protein activity (cont)Some loss-of-function mutations can be dominant-negativeUsually occurs in genes that encode multimeric proteinsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.31bMutant subunits block the activity of normal subunitsFig. 8.31cKinky allele of fused locusMultimeric protein made of four subunitsGain-of-function mutations enhance a function or confer a new activityGain-of-function mutations are usually dominantHypermorphic mutations – generate more gene product or the same amount of a more efficient gene productNeomorphic mutations – generate gene product with new function or that is expressed at inappropriate time or placeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8*Fig. 8.31Mutation in Antennapedia gene of Drosophila causes ectopic expression of a leg-determining gene in structures that normally produce antennaeMutantWild-typeMutations classified by their effects on protein function*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Table 8.2The cellular components of gene expressionMutations in genes encoding gene products for transcription, RNA processing, translation, and protein processing are often lethalSome mutations in tRNA genes can suppress mutations in protein-coding genes*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Table 8.3A nonsense mutation in a protein-coding gene creates a truncated, nonfunctional protein*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig 8.32aNonsense suppressionA second, nonsense suppressing mutation in the anticodon of a tRNA gene allows production of a (mutant) full-length polypeptide*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8Fig 8.32b