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Unformatted text preview: 0 Gene Expression
The Flow of Genetic Information from DNA via RNA to Protein Chapter 8 I. The genetic code: how precise groupings of the 4 nucleotides specify 20 amino acids A. In the genetic code, a triplet represents each amino acid B. Mapping studies confirm that gene's nucleotide sequence corresponds to the actual amino acid sequence C. Genetic analysis revealed that NON-overlapping codons are set in a specific reading frame 1. codon is composed of more than one nucleotide 2. each nucleotide is part of only ONE codon 3. codons = 3 nucleotides; designated start point for each gene establishes the proper reading frame 0 D. Cracking the code: certain codons specify certain amino acids 1. discovery of mRNA, molecules for transporting genetic info. 2. synthetic mRNAs made it possible to discover which codons designate which amino acids 3. the 5'- to - 3' direction of mRNA corresponds to the N-terminal and C-terminal direction in a polypeptide 4. nonsense codons cause termination of a polypeptide chain E. Genetic code: A summary F. Using genetics to verify the code G. Genetic code is almost, but not quite, universal II. Transcription: RNA polymerase synthesizes a mRNA copy of the gene's template A. Details of the process B. Eukaryotes: mRNA processing after transcription produces a mature mRNA 1. adding a methylated cap at the 5' end and poly-A tail at the 3' end 2. RNA splicing removes sequences known as introns from the primary transcript 3. alternative splicing often produces different mRNAs from the same primary transcript III. Translation: base-pairing between mRNA and tRNAs directs assembly of a polypeptide on the ribosome A. tRNAs mediate the translation of of mRNA codons to amino acids 1. tRNA structure: a compact "L" carrying an anticodon at one end and an amino acid at the other 2. base-pairing between a mRNA codon and a tRNA anticodon directs incorporation of an amino acid into a growing polypeptide 3. Wobble: some tRNAs can recognize more than one codon B. Ribosomes are the sight of polypeptide synthesis 1. complex structures composed of RNA and protein 2. different parts of the ribosome have different functions C. Mechanism of translation: differences between eukaryotes and prokaryotes D. Processing after translation can change a polypeptide's structure IV. Comprehensive example: a computerized analysis of gene expression in C. elegans V. How mutations affect gene expression A. Mutations in a gene's coding sequence can alter the gene product 1. silent mutations do not alter the amino acid specified 2. missense mutations replace one amino acid with another 3. nonsense mutations introduce a STOP codon 4. frameshift mutations result from the insertion or deletion of nucleotides within the coding sequence B. Mutations in a gene outside the coding sequence can also alter gene expression C. Mutations in genes encoding the molecules that implement expression may effect transcription, mRNA splicing, or translation 1. mutations altering genes encoding proteins or RNAs involved in gene expression are usually lethal 2. mutations in tRNA genes can suppress mutations in proteincoding genes The triplet codon represents each amino acid 0 20 amino acids encoded for by 4 nucleotides By deduction:
1 nucleotide/amino acid = 41 = 4 combinations. 2 nucleotides/amino acid = 42 = 16 combinations. 3 nucleotides/amino acid = 43 = 64 combinations. Must be at least triplet combinations that code for amino acids The Genetic Code: 61 triplet codons represent 020 amino acids; 3 triplet codons signify stop No need to memorize this table, but you should be able to APPLY it Fig. 8.3 A gene's nucleotide sequence is co-linear with the amino acid sequence of the encoded polypeptide Charles Yanofsky E. coli genes for a subunit of tyrptophan synthetase compared mutations within a gene to particular amino acid substitutions. Created Trp- mutants in trpA Created fine structure recombination map Determined amino acid sequences of mutants and was able to determine several results A codon is composed of more than one nucleotide.
Different point mutations may affect same amino acid. Codon contains more than one nucleotide. Each nucleotide is part of only a single codon. Each point mutation altered only one amino acid. A codon is composed of three nucleotides and the starting point of each gene establishes a reading frame
studies of frameshift mutations in bacteriophage T4 rIIB gene Fig. 8.5 Most amino acids are specified by more than one codon. Phenotypic effect of frameshifts depends on if reading frame is restored. Fig. 8.6 Cracking the code: biochemical manipulations revealed which codons represent which amino acids The discovery of messenger RNAs, molecules for transporting genetic information Protein synthesis takes place in cytoplasm deduced from radioactive tagging of amino acids. RNA, an intermediate molecule made in nucleus and transports DNA information to cytoplasm Synthetic mRNAs and in vitro translation determines which codons designate which amino acids 1961 Marshall Nirenberg and Heinrich Matthaei created mRNAs and translated to polypeptides in vitro Polymononucleotides Polydinucleotides Polytrinucleotides Polytetranucleotides Read amino acid sequence and deduced codons Fig. 8.7 Ambiguities resolved by Nirenberg and Philip Leder using trinucleotide mRNAs of known sequence to tRNAs charged with radioactive amino acid with ribosomes Fig. 8.8 5' to 3' direction of mRNA corresponds to N-terminal-to-Cterminal direction of polypeptide. One strand of DNA is a template. The other is an RNA-like strand (non-template strand). Nonsense codons cause termination of a polypeptide chain UAA (ocher), UAG (amber), and UGA (opal). Fig. 8.9 Codon consist of a triplet codon each of which specifies an amino acid. Summary Code shows a 5' to 3' direction. Codons are non-overlapping. Code includes three stop codons, UAA, UAG, and UGA that terminate translation. Code is degenerate. Fixed starting point establishes a reading frame. AUG in an initiation codon which specifies reading frame. 5'- 3' direction of mRNA corresponds with N-terminus to Cterminus of polypeptide. Mutation modify message encoded in sequence Frameshift mutations change reading frame. Missense mutations change codon of amino acid to another amino acid. Nonsense mutations change a codon for an amino acid to a stop codon. Do living cells construct polypeptides according to same rules as in vitro experiments? Studies of how mutations affect amino-acid composition of polypeptides encoded by a gene Missense mutations induced by mutagens should be single nucleotide substitutions and conform to the code. Fig. 8.10 a Genetic code is almost universal but not quite All living organisms use same basic genetic code.
Translational systems can use mRNA from another organism to generate protein. Comparisons of DNA and protein sequence reveal perfect correspondence between codons and amino acids among all organisms. Transcription RNA polymerase catalyzes transcription. Promoters signal RNA polymerase where to begin transcription. RNA polymerase adds nucleotides in 5' to 3' direction. Terminator sequences tell RNA when to stop transcription. (UAA, UAG,UGA) Initiation of transcription Fig. 8.11 a Elongation Fig. 8.11 b Termination Fig. 8.11 c Information flow Fig. 8.11 d Promoters of 10 different bacterial genes Fig. 8.12 In eukaryotes, RNA is processed after transcription A 5' methylated cap and a 3' Poly-A tail are added. Structure of the methylated cap How Poly-A tail is added to 3' end of mRNA Fig. 8.14 RNA splicing removes introns Exons 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 introns. Dystrophin gene underlying Duchenne muscular dystrophy (DMD) is an extreme example of introns Fig. 8.15 How RNA processing splices out introns and adjoins adjacent exons Fig. 8.16 Splicing is catalyzed by spliceosomes.
Ribozymes RNA molecules that act as enzymes Ensures that all splicing reactions take place in concert Fig. 8.17 Alternative splicing
Different mRNAs can be produced by same transcript. Rare transplicing events combine exons from different genes. Fig. 8.18 Translation Transfer RNAs (tRNAs) mediate translation of mRNA codons to amino acids. tRNAs carry anticodon on one end. Three nucleotides complementary to an mRNA codon Primary nucleotide sequence Secondary short complementary sequences pair and make clover leaf shape Tertiary folding into three dimensional space shape like an L Structure of tRNA Base pairing between an mRNA codon and a tRNA anticodon directs amino acid incorporation into a growing polypeptide. Charged tRNA is covalently coupled to its amino acid. Many tRNAs contain modified bases Fig. 8.19 a Secondary and tertiary structure Fig. 8.19 b Aminoacyl-tRNA synthetase catalyzes attachment of tRNAs to corresponding amino acid Fig. 8.20 Base pairing between mRNA codon and tRNA anticodon determines where incorporation of amino acid occurs Fig. 8.21 Wobble: Some tRNAs recognize more than one codon for amino acids they carry. Fig. 8.22 Rhibosomes are site of polypeptide synthesis Ribosomes are complex structures composed of RNA and protein. Fig. 8.23 Initiation sets stage for polypeptide synthesis. Mechanism of translation AUG start codon at 5' end of mRNA Formalmethionine (fMet) on initiation tRNA First amino acid incorporated in bacteria Elongation during which amino acids are added to growing polypeptide Ribosomes move in 5'-3' direction revealing codons. Addition of amino acids to C terminus 2-15 amino acids per second Nonsense codon recognized at 3' end of reading frame Release factor proteins and halt polypeptide synthesis Termination which halts polypeptide synthesis Initiation of translation Fig. 8.24 a Elongation Fig. 8.24 b Termination of translation Fig. 8.24 c Posttranslational processing can modify polypeptide structure. Fig. 8.25 Significant differences in gene expression between prokaryotes and eukaryotes Eukaryotes, nuclear membrane prevents coupling of transcription and translation. Prokaryotic messages are polycistronic. Contain information for multiple genes Eukaryotes, small ribosomal subunit binds to 5' methylated cap and migrates to AUG start codon. 5' untranslated leader sequence between 5' cap and AUG start Only a single polypeptide produced from each gene Initiating tRNA in prokaryotes is fMet. Initiating tRNA in eukaryotes Met is unmodified. A computerized analysis of gene expression in C. elegans: A comprehensive example Computer programs search for possible exons by looking for strings of codons uninterrupted by nonsense codons. Look for splice donor and acceptor sites to identify introns. C. elegans genome contains roughly 19,000 genes. 15% encode worm's genes or proteins. Landmarks in a callogen gene of C. elegans and comparison of DNA and mRNA sequences
Fig. 8.26 Mutations in a gene's coding sequence can alter the gene product. Silent mutations do not alter amino acid specified. Missense mutations replace one amino acid with another. Nonsense mutations change an amino-acid-specifying codon to a stop codon. Frameshift mutations result from the insertion or deletion of nucleotides within the coding sequence. Mutations outside of the coding sequence can also alter gene expression. Promoter sequences Termination signals Splice-acceptor and splice-donor sites Ribosome binding sites Fig. 8.27 c Mutations in genes encoding the molecules that implement expression may affect transcription, l mRNA splicing, or translation.
Usually lethal Mutations in tRNA genes can suppress mutations in protein-coding genes. Nonsense suppressor tRNAs Nonsense suppression (a) Nonsense mutation that causes incomplete nonfunctional polypeptide (b) Nonsensesuppressing mutation causes addition of amino acid at stop codon allowing production of full length polypeptide. Fig. 8.28 ...
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