1、Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,8-1,Prepared by Malcolm Schug University of North Carolina Greensboro,8-2,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Gene Expression,The Flow of Genetic Information from D
2、NA via RNA to Protein,8-3,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Outline of Chapter 8,The genetic code How triplets of the four nucleotides unambiguously specify 20 amino acids, making it possible to translate information from a nucleotide chain to a se
3、quence of amino acids Transcription How RNA polymerase, guided by base pairing, synthesizes a single-stranded mRNA copy of a genes DNA template Translation How base pairing between mRNA and tRNAs directs the assembly of a polypeptide on the ribosome Significant differences in gene expression between
4、 prokaryotes and eukaryotes How mutations affect gene information and expression,8-4,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,The triplet codon represents each amino acid.,20 amino acids encoded for by 4 nucleotides By deduction: 1 nucleotide/amino acid =
5、 41 = 4 triplet combinations. 2 nucleotides/amino acid = 42 = triplet combinations. 3 nucleotides/amino acid = 43 = triplet combinations. Must be at least triplet combinations that code for amino acids,8-5,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,The Gene
6、tic Code: 61 triplet codons represent 20 amino acids; 3 triplet codons signify stop.,Fig. 8.3,8-6,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,A genes nucleotide sequence is colinear the amino acid sequence of the encoded polypeptide.,Charles Yanofsky E. coli
7、 genes for a subunit of tyrptophan synthetase compared mutations within a gene to particular amino acid substitutions. Trp- mutants in trpA Fine structure recombination map Determined amino acid sequences of mutants,8-7,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or di
8、splay,Fig. 8.4,8-8,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,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. Eac
9、h point mutation altered only one amino acid.,8-9,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,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 g
10、ene,Fig. 8.5,8-10,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Most amino acids are specified by more than one codon. Phenotypic effect of frameshifts depends on if reading frame is restored.,Fig. 8.6,8-11,Copyright The McGraw-Hill Companies, Inc. Permission
11、required to reproduce or display,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.
12、 RNA, an intermediate molecule made in nucleus and transports DNA information to cytoplasm,8-12,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Synthetic mRNAs and in vitro translation determines which codons designate which amino acids.,1961 Marshall Nirenberg
13、and Heinrich Mathaei created mRNAs and translated to polypeptides in vitro Polymononucleotides Polydinucleotides Polytrinucleotides Polytetranucleotides Read amino acid sequence and deduced codons,Fig. 8.7,8-13,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Amb
14、iguities resolved by Nirenberg and Philip Leder using trinucleotide mRNAs of known sequence to tRNAs charged with radioactive amino acid with ribosomes,Fig. 8.8,8-14,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,5 to 3 direction of mRNA corresponds to N-termin
15、al-to-C-terminal direction of polypeptide. One strand of DNA is a template. The other is an RNA-like strand. Nonsense codons cause termination of a polypeptide chain UAA (ocher), UAG (amber), and UGA (opal).,Fig. 8.9,8-15,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or
16、display,Summary,Codon consist of a triplet codon each of which specifies an amino acid. Code shows a 5 to 3 direction. Codons are nonoverlapping. Code includes three stop codons, UAA, UAG, and UGA that terminate translation. Code is degenerate. Fixed starting point establishes a reading frame. UAG i
17、n an initiation codon which specifies reading frame. 5- 3 direction of mRNA corresponds with N-terminus to C-terminus of polypeptide. Mutaiton modify message encoded in sequence Frameshift mutaitons change reading frame. Missense mutations change codon of amino acid to another amino acid. Nonsense m
18、utations change a codon for an amino acid to a stop codon.,8-16,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Do living cells construct polypeptides according to same rules as in vitro experiments?,Studies of how mutations affect amino-acid composition of poly
19、peptides encoded by a gene Missense mutations induced by mutagens should be single nucleotide substitutions and conform to the code.,Fig. 8.10 a,8-17,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Proflavin treatment generates trp- mutants. Further treatment ge
20、nerates trp+ revertants. Single base insertion (trp-) and a deletion causes reversion (trp+).,Fig. 8.10 b,8-18,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Genetic code is almost universal but not quite.,All living organisms use same basic genetic code. Trans
21、lational 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.,8-19,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Transcription,
22、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.,8-20,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or disp
23、lay,Initiation of transcription,Fig. 8.11 a,8-21,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Elongation,Fig. 8.11 b,8-22,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Termination,Fig. 8.11 c,8-23,Copyright The McGraw-H
24、ill Companies, Inc. Permission required to reproduce or display,Information flow,Fig. 8.11 d,8-24,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Promoters of 10 different bacterial genes,Fig. 8.12,8-25,Copyright The McGraw-Hill Companies, Inc. Permission requir
25、ed to reproduce or display,In eukaryotes, RNA is processed after transcription.,A 5 methylated cap and a 3 Poly-A tail are added. Structure of the methylated cap,Fig. 8.13,8-26,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,How Poly-A tail is added to 3 end of
26、mRNA,Fig. 8.14,8-27,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,RNA splicing removes introns.,Exons sequences found in a genes DNA and mature mRNA (expressed regions) Introns sequences found in DNA but not in mRNA (intervening regions) Some eukaryotic genes
27、have many introns.,8-28,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Dystrophin gene underlying Duchenne muscular dystrophy (DMD) is an extreme example of introns.,Fig. 8.15,8-29,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or di
28、splay,How RNA processing splices out introns and adjoins adjacent exons,Fig. 8.16,8-30,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Splicing is catalyzed by spliceosomes. Ribozymes RNA molecules that act as enzymes Ensures that all splicing reactions take pla
29、ce in concert,Fig. 8.17,8-31,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Alternative splicing Different mRNAs can be produced by same transcript. Rare transplicing events combine exons from different genes.,Fig. 8.18,8-32,Copyright The McGraw-Hill Companies,
30、 Inc. Permission required to reproduce or display,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 Structure of tRNA Primary nucleotide sequence Secondary short complementary sequen
31、ces pair and make clover leaf shape Tertiary folding into three dimensional space shape like an L 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.,8-33,Copyright The McGraw-Hill
32、Companies, Inc. Permission required to reproduce or display,Many tRNAs contain modified bases.,Fig. 8.19 a,8-34,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Secondary and tertiary structure,Fig. 8.19 b,8-35,Copyright The McGraw-Hill Companies, Inc. Permission
33、 required to reproduce or display,Aminoacyl-tRNA syntetase catalyzes attachment of tRNAs to corresponding amino acid.,Fig. 8.20,8-36,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Base pairing between mRNA codon and tRNA anticodon determines where incorporation
34、 of amino acid occurs.,Fig. 8.21,8-37,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Wobble: Some tRNAs recognize more than one codon for amino acids they carry.,Fig. 8.22,8-38,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displa
35、y,Rhibosomes are site of polypeptide synthesis.,Ribosomes are complex structures composed of RNA and protein.,Fig. 8.23,8-39,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Mechanism of translation,Initiation sets stage for polypeptide synthesis. AUG start codon
36、 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 Termination
37、which halts polypeptide synthesis Nonsense codon recognized at 3 end of reading frame Release factor proteins and halt polypeptide synthesis,8-40,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Initiation of translation,Fig. 8.25 a,8-41,Copyright The McGraw-Hill
38、 Companies, Inc. Permission required to reproduce or display,Elongation,Fig. 8.25 b,8-42,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Termination of translation,Fig. 8.25 c,8-43,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or dis
39、play,Posttranslational processing can modify polypeptide structure.,Fig. 8.26,8-44,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Significant differences in gene expression between prokaryotes and eukaryotes,Eukaryotes, nuclear membrane prevents coupling of tra
40、nscription 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
41、 each gene Initiating tRNA in prokaryotes is fMet. Initiating tRNA in eukaryotes Met is unmodified.,8-45,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,8-46,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,A computerized ana
42、lysis 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 wor
43、ms genes or proteins.,8-47,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Landmarks in a callogen gene of C. elegans and comparison of DNA and mRNA sequences,Fig. 8.27,8-48,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Mu
44、tations in a genes coding sequence can alter the gene product. 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. Silen
45、t mutations do not alter amino acid specified.,Fig. 8.28,8-49,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Mutations outside of the coding sequence can also alter gene expression. Promoter sequences Termination signals Splice-acceptor and splice-donor sites R
46、ibosome binding sites,Fig. 8.28 b,8-50,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Loss-of-function alleles are usually recessive.,Null or amorphic mutations are alleles that completely block the function of a protein. Hypomorphic mutations produce much less
47、 of a protein or a protein with weak but detectable function.,Rocket immunoelectrophoresis reveals the amount of xanthine dehydrogenase produced in flies with different genotypes. Null allele 1 and hypomorphic allele 2 are recessive to wildtype.,Fig. 8.29,8-51,Copyright The McGraw-Hill Companies, In
48、c. Permission required to reproduce or display,Incomplete dominance arises when phenotype varies in proportion to the amount of protein.,8-52,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Rarely, loss-of-function mutations are dominant.,Haploinsufficiency one
49、wild-type allele does not provide enough of a gene product,Heterozygotes for the null mutation of the T locus in mice have short tails because they have an insufficient amount of protein to produce a wild-type tail.,Fig. 8.31 a,8-53,Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display,Dominant-negative mutations alleles of a gene encoding subunits of multimers that block the activity of subunits produced by normal alleles,
