Concepts of Genetics PDF - Chapter 13 - The Genetic Code and Transcription

3/24/2020 Concepts of Genetics Twelfth Edition, Global Edition Chapter 13 The Genetic Code and Transcription...

3/24/2020 Concepts of Genetics Twelfth Edition, Global Edition Chapter 13 The Genetic Code and Transcription Lecture Presentation by Dr. Cindy Malone California State University Northridge Copyright © 2019 Pearson Education Ltd. All Rights Reserved Learning Objectives (1 of 2) 13.1 The Genetic Code Uses Ribonucleotide Bases as “Letters” 13.2 Early Studies Established the Basic Operational Patterns of the Code 13.3 Studies by Nirenberg, Matthaei, and Others Led to Deciphering of the Code 13.4 The Coding Dictionary Reveals Several Interesting Patterns among the 64 Codons 13.5 The Genetic Code Has Been Confirmed in Studies of Phage MS2 Copyright © 2019 Pearson Education Ltd. All Rights Reserved 1  3/24/2020 Learning Objectives (2 of 2) 13.6 The Genetic Code Is Nearly Universal 13.7 Different Initiation Points Create Overlapping Genes 13.8 Transcription Synthesizes RNA on a DNA Template 13.9 RNA Polymerase Directs RNA Synthesis 13.10 Transcription in Eukaryotes Differs from Bacterial Transcription in Several Ways 13.11 The Coding Regions of Eukaryotic Genes Are Interrupted by Intervening Sequences Called Introns 13.12 RNA Editing May Modify the Final Transcript 13.13 Transcription Has Been Visualized by Electron Microscopy Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.1 Introduction (1 of 2) The central dogma of molecular genetics – The directional flow of genetic information from DN A to RNA to protein Copyright © 2019 Pearson Education Ltd. All Rights Reserved 2  3/24/2020 13.1 The Genetic Code Uses Ribonucleotide Bases as “Letters” Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.1 The Genetic Code (1 of 3) The genetic code: General features – Written in linear form using ribonucleotide bases that compose mRNA – Each “word” consists of three ribonucleotide letters, or a triplet code ▪ Codon: Every three ribonucleotides – Unambiguous—each triplet specifies only one amino acid Copyright © 2019 Pearson Education Ltd. All Rights Reserved 3  3/24/2020 Section 13.1 The Genetic Code (2 of 3) The genetic code – Degenerate: A given amino acid can be specified by more than one triplet codon – Contains “start” and “stop” signals: triplets that initiate and terminate translation – Commaless: Once translation begins, codons are read with no break Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.1 The Genetic Code (3 of 3) – Nonoverlapping: Any single ribonucleotide within mRNA is part of one triplet – Colinear: Sequence of codons in a gene is colinear – Nearly universal: A single coding dictionary is used by viruses, prokaryotes, archaea, and eukaryotes Copyright © 2019 Pearson Education Ltd. All Rights Reserved 4  3/24/2020 13.2 Early Studies Established the Basic Operational Patterns of the Code Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.2 Operational Patterns mRNA—messenger RNA – Serves as intermediate in transferring genetic information from DNA to proteins – Genetic information is stored in DNA – Code that translates it to protein is in RNA Copyright © 2019 Pearson Education Ltd. All Rights Reserved 5  3/24/2020 Section 13.2 The Triplet Code Triplet code – Provides 64 codons to specify 20 amino acids Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.2 Frameshift Mutations (1 of 2) Reading frame – Contiguous sequence of nucleotides – Insertions or deletions shift reading frame and change codons downstream = frameshift mutation Triplet nature code was revealed by frameshift mutations Copyright © 2019 Pearson Education Ltd. All Rights Reserved 6  3/24/2020 Section 13.2 Frameshift Mutations (2 of 2) Figure 13.2 The effect of frameshift mutations on a DNA sequence with the repeating triplet sequence GAG. (a) The insertion of a single nucleotide shifts all subsequent triplet reading frames. (b) The insertion of three nucleotides changes only two triplets, but the frame of reading is then re-established to the original sequence. Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.2 Nonoverlapping Code Nonoverlapping – Genetic code reads three nucleotides at a time in continuous, linear manner – During translation, genetic code is nonoverlapping Copyright © 2019 Pearson Education Ltd. All Rights Reserved 7  3/24/2020 13.3 Studies by Nirenberg, Matthaei, and Others Led to Deciphering of the Code Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.3 Polynucleotide Phosphorylase Synthetic polypeptides Polynucleotide phosphorylase – Enzyme that catalyzes production of synthetic mRNAs – mRNAs serve as template for in vitro system ▪ cell-free in test tube Figure 13.3 The reaction catalyzed by the enzyme polynucleotide phosphorylase. Note that the equilibrium of the reaction favors the degradation of RNA but that the reaction can be “forced” in the direction favoring synthesis. Copyright © 2019 Pearson Education Ltd. All Rights Reserved 8  3/24/2020 Section 13.3 RNA Homopolymers RNA homopolymers – RNA nucleotides with only one type of ribonucleotide – RNA homopolymers were added to in vitro translation system – Helped decipher which amino acids were encoded by first few codons based on which amino acids were incorporated into polypeptide Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.3 RNA Heteropolymers (1 of 2) RNA heteropolymers – Two or more different ribonucleosides were used to decipher relative proportion of each type of ribonucleotide diphosphate in synthetic mRNA Copyright © 2019 Pearson Education Ltd. All Rights Reserved 9  3/24/2020 Section 13.3 RNA Heteropolymers (2 of 2) Figure 13.4 Results and interpretation of a heteropolymer experiment in which a ratio of 1A : 5C (1/6A : 5/6C) is used. Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.3 Triplet Binding Assay (1 of 3) Triplet binding assay – Developed by Nirenberg and Leder to determine other specific codon assignments – Ribosomes bind to single codon of three nucleotides – Complementary amino acid-charged tRNA can bind Copyright © 2019 Pearson Education Ltd. All Rights Reserved 10  3/24/2020 Section 13.3 Triplet Binding Assay (3 of 3) Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.3 Repeating Copolymers (1 of 3) Chemically synthesized long RNAs – Short repeating sequences enzymatically joined short sequences together, which made long RNAs – Used for in vitro translation to determine more codon assignments Copyright © 2019 Pearson Education Ltd. All Rights Reserved 11  3/24/2020 Section 13.3 Repeating Copolymers (2 of 3) Figure 13.6 The conversion of di-, tri-, and tetranucleotides into repeating copolymers. The triplet codons produced in each case are shown. Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.3 Repeating Copolymers (3 of 3) Table 13.3 Amino Acids Incorporated Using Repeated Synthetic Copolymers of RNA Copyright © 2019 Pearson Education Ltd. All Rights Reserved 12  3/24/2020 13.4 The Coding Dictionary Reveals Several Interesting Patterns among the 64 Codons Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.4 Degeneracy of the Genetic Code (1 of 2) The genetic code is degenerate – Many amino acids specified by more than one codon – Only tryptophan and methionine are encoded by single codon – Genetic code shows order: ▪ Chemically similar amino acids share one or two middle bases in triplets encoding them Copyright © 2019 Pearson Education Ltd. All Rights Reserved 13  3/24/2020 Section 13.4 Degeneracy of the Genetic Code (2 of 2) Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.4 The Wobble Hypothesis (1 of 2) The wobble hypothesis – The initial two ribonucleotides of triplet codes are often more critical than the third – Third position ▪ Less spatially constrained ▪ Need not adhere as strictly to established base- pairing rules Copyright © 2019 Pearson Education Ltd. All Rights Reserved 14  3/24/2020 Section 13.4 The Wobble Hypothesis (2 of 2) Table 13.4 Anticodon–Codon Base-Pairing Rules Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.4 Initiation Methionine (AUG)—initiator codon – Initial amino acid incorporated into all proteins – In bacteria: modified form of methionine ▪ N-formylmethionine (fmet) AUG: Only codon to encode methionine – Appears internally in mRNA unformylated Copyright © 2019 Pearson Education Ltd. All Rights Reserved 15  3/24/2020 Section 13.4 Termination Termination codons: UAG, UAA, UGA – Do not code for any amino acid – Are not recognized by tRNA – Translation terminates when these codons are encountered Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.4 Nonsense Mutations Nonsense mutations – Mutations that produce a stop codon internally in gene – Translation is terminated – Partial polypeptide is produced Copyright © 2019 Pearson Education Ltd. All Rights Reserved 16  3/24/2020 13.5 The Genetic Code Has Been Confirmed in Studies of Phage MS2 Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.5 Phage MS2 Phage MS2 – Bacteriophage that infects E. coli – Phage RNA contains only three genes on 3500-base RNA genome – Genes specify coat protein, RNA-directed replicase, and maturation protein – Sequencing gene products confirmed genetic code Copyright © 2019 Pearson Education Ltd. All Rights Reserved 17  3/24/2020 13.6 The Genetic Code is Nearly Universal Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.6 Mitochondrial DNA (mtDNA) Mitochondrial DNA – Revealed exceptions to universal genetic code – Codon UGA normally specifies termination ▪ mtDNA UGA codon encodes tryptophan in yeast and humans – Codon AUA normally specifies isoleucine ▪ Human mitochondria encodes internal insertion of methionine Copyright © 2019 Pearson Education Ltd. All Rights Reserved 18  3/24/2020 Section 13.6 Exceptions to Universal Genetic Code Copyright © 2019 Pearson Education Ltd. All Rights Reserved 13.7 Different Initiation Points Create Overlapping Genes Copyright © 2019 Pearson Education Ltd. All Rights Reserved 19  3/24/2020 Section 13.7 Overlapping Genes Overlapping genes )‫(الجينات المتداخلة‬ – Single mRNA has multiple initiation points – Creates different reading frames (‫)إطارات القراءة‬ – Specifies more than one polypeptide Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.7 Open Reading Frames (1 of 2) ORF: Open reading frame (overlapping genes) – DNA sequence produces RNA with start and stop – Series of triplet codons specify amino acids to make polypeptide In some viruses, initiation at different AUG positions out of frame with another leads to distinct polypeptides Copyright © 2019 Pearson Education Ltd. All Rights Reserved 20  3/24/2020 Section 13.7 Open Reading Frames (2 of 2) Figure 13.8 Illustration of the concept of overlapping reading frames. (a) Translation initiated at two different AUG positions out of frame with one another will give rise to two distinct amino acid sequences. (b) The relative positions of the sequences encoding seven polypeptides of the phage fX174. Copyright © 2019 Pearson Education Ltd. All Rights Reserved 13.8 Transcription Synthesizes RNA on a DNA Template Copyright © 2019 Pearson Education Ltd. All Rights Reserved 21  3/24/2020 Section 13.8 Transcription Transcription – RNA synthesized on DNA template – Genetic information stored in DNA is transferred to RN A – Serves as intermediate molecule between DNA and proteins – Each triplet codon is complementary to anticodon of t RN A Copyright © 2019 Pearson Education Ltd. All Rights Reserved 13.9 RNA Polymerase Directs RNA Synthesis Copyright © 2019 Pearson Education Ltd. All Rights Reserved 22  3/24/2020 Section 13.9 RNA Polymerase RNA polymerase – Enzyme directs synthesis of RNA using DNA template – Nucleotides contain ribose, not deoxyribose – No primer required for initiation n(NTP)  DNA RNA polymerase (NMP)n  n(PPi ) Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.9 Promoters (1 of 2) Transcription results in ssRNA – Template strand is transcribed – Transcription begins with template binding by RNA polymerase at promoter – Promoters: Specific DNA sequences in 5′ region upstream of initial transcription point – σ subunit responsible for promoter recognition (initiation of transcription) Copyright © 2019 Pearson Education Ltd. All Rights Reserved 23  3/24/2020 Section 13.9 Promoters (2 of 2) Figure 13.9 The early stages of transcription in bacteria, showing (a) the components of the process; (b) template binding at the –10 site involving the sigma subunit of RNA polymerase and subsequent initiation of RNA synthesis; and (c) chain elongation, after the subunit has dissociated from the transcription complex and the enzyme moves along the DNA template. Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.9 Transcription Start Site Transcription start site – DNA double helix is denatured: unwound to make template strand accessible for RNA polymerase – Interaction of promoters and RNA polymerase regulates efficiency of transcription Copyright © 2019 Pearson Education Ltd. All Rights Reserved 24  3/24/2020 Section 13.9 Consensus Sequences Consensus sequences – DNA sequences homologous in different genes of same organism E. coli promoters have two consensus sequences – TTGACA and TATAAT (Pribnow box) – Positioned at −35 and −10 with respect to the transcription initiation site Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.9 Chain Elongation Chain elongation – Ribonucleotides are added to RNA chain – σ subunit dissociates from holoenzyme – Elongation proceeds under direction of core enzyme Copyright © 2019 Pearson Education Ltd. All Rights Reserved 25  3/24/2020 Section 13.9 Promoters Figure 13.9 The early stages of transcription in bacteria, showing (a) the components of the process; (b) template binding at the –10 site involving the sigma subunit of RNA polymerase and subsequent initiation of RNA synthesis; and (c) chain elongation, after the s subunit has dissociated from the transcription complex and the enzyme moves along the DNA template. Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.9 Termination (1 of 2) Termination – Enzyme traverses entire gene until a termination nucleotide sequence is encountered – In bacteria: Termination transcribed into RNA causes newly formed transcript to fold back on itself (hairpin) – Rho-dependent termination depends on the rho (ρ) termination factor Copyright © 2019 Pearson Education Ltd. All Rights Reserved 26  3/24/2020 Section 13.9 Termination (2 of 2) Figure 13.10 Transcription termination in bacteria. Intrinsic termination (a) involves a hairpin structure followed by a string of repeated U residues. Rho- dependent termination (b) involves the termination factor rho and a hairpin structure. Copyright © 2019 Pearson Education Ltd. All Rights Reserved 13.10 Transcription in Eukaryotes Differs from Prokaryotic Transcription in Several Ways Copyright © 2019 Pearson Education Ltd. All Rights Reserved 27  3/24/2020 Section 13.10 Eukaryotic Transcription Transcription in eukaryotes – Occurs within nucleus (unlike prokaryotes) – mRNA must leave nucleus for translation – Chromatin remodeling: Chromatin must uncoil to make DNA accessible to RNA Pol – RNA polymerases rely on transcription factors (TFs) to scan/bind DNA – Enhancers and silencers control transcription regulation Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.10 RNA Polymerases (1 of 2) Eukaryotes possess three forms of RNA polymerase – Each transcribes different types of genes – RNA Pol I – RNA Pol II – RNA Pol III Copyright © 2019 Pearson Education Ltd. All Rights Reserved 28  3/24/2020 Section 13.10 RNA Pol II RNA polymerase II (RNAP II) – Responsible for transcription of wide range of genes in eukaryotes – RNAP II core-promoter determines where RNAP II binds to DNA Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.10 TATA Box Regulatory sequences influence efficiency of transcription initiation by RNAP II – Proximal-promoter elements – Enhancers – Silencers TATA box – Core-promoter element – Binds TATA-binding protein (TBP) of transcription factor TFIID: determines start transcription start site Copyright © 2019 Pearson Education Ltd. All Rights Reserved 29  3/24/2020 Section 13.10 Enhancers and Silencers Enhancers and silencers – Found upstream, within, or downstream of gene – Enhancers increase transcription levels; silencers decrease them – Modulate transcription from a distance – Act to increase or decrease transcription in response to cell’s requirement for gene product Copyright © 2019 Pearson Education Ltd. All Rights Reserved Insulator (‫ )عازل‬is the name given to a class of DNA sequence elements that possess a common ability to protect genes from inappropriate signals emanating from their surrounding environment. Enhancers short region of DNA (50- 1500 bp) that can be found by proteins (activators) to increase the likelihood that a particular gene will be transcribed. Activators are transcription factors. Silencers are antagonists of enhancers , when bound to TF( repressors), they stop transcription of the gene. Repressors are another type of transcription factors. Copyright © 2019 Pearson Education Ltd. All Rights Reserved 30  3/24/2020 Section 13.10 Transcription Factors Transcription factors facilitate RNAP II binding and initiation of transcription – General transcription factors: Required for all RNAP II-mediated transcription – Transcription activators and repressors: Influence efficiency or rate of RNAP II transcription initiation Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.10 Cap and Tail Eukaryotic mRNAs require processing to produce mature mRNAs Posttranscriptional modifications – Addition of 5′ cap (7-mG cap) – Addition of 3′ tail (poly-A tail) – Excision of introns Copyright © 2019 Pearson Education Ltd. All Rights Reserved 31  3/24/2020 Section 13.10 RNA Processing Figure 13.11 Posttranscriptional RNA processing in eukaryotes. Beginning at the promoter (P) of a gene, transcription produces a pre-mRNA containing several introns (I) and exons (E), as identified under the DNA template strand. Shortly after transcription begins, a m7G cap is added to the 5’ end. Next, and during transcription elongation, the introns are spliced out and the exons joined. Finally, a poly-A tail is added to the 3’ end. While this figure depicts these steps sequentially, in some eukaryotic transcripts, the poly-A tail is added before splicing of all introns has been completed. Copyright © 2019 Pearson Education Ltd. All Rights Reserved Animation: Transcription Copyright © 2019 Pearson Education Ltd. All Rights Reserved 32  3/24/2020 13.11 The Coding Regions of Eukaryotic Genes Are Interrupted by Intervening Sequences Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.11 Introns and Exons Introns (intervening sequences) – Regions of initial RNA transcript not expressed in amino acid sequence of protein – DNA sequences not represented in final mRNA product – Exons are sequence retained and expressed – Prokaryotes do not have introns – Heteroduplexes: Introns present in DNA but not mRNA loop out Copyright © 2019 Pearson Education Ltd. All Rights Reserved 33  3/24/2020 Section 13.11 Splicing Posttranscriptional modification: Splicing – Introns are removed by splicing – Exons are then joined together in mature mRNA – Mature mRNA is smaller than initial RNA Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.11 Introns and Exons (1 of 2) Figure 13.12 Intron and exon sequences in various eukaryotic genes. The numbers indicate the number of nucleotides present in various intron and exon regions. Copyright © 2019 Pearson Education Ltd. All Rights Reserved 34  3/24/2020 Section 13.11 Introns and Exons (2 of 2) Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.11 Self-Splicing RNAs and Spliceosome (1 of 4) Self-Splicing RNAs – Self-excision group I introns occurs in bacteria, lower eukaryotes, and higher plants https://www.youtube.com/watch?v=nUhChbEBC3c Figure 13.13 Splicing mechanism for removal of a group I intron. The process is one of self-excision involving two transesterification reactions. Copyright © 2019 Pearson Education Ltd. All Rights Reserved 35  3/24/2020 Section 13.11 Self-Splicing RNAs and Spliceosome (3 of 4) Spliceosome – Pre-mRNA introns spliced out by spliceosome – Reaction involves: ▪ Formation of lariat structure ▪ Splice donor and acceptor sites ▪ Branch point sequence Figure 13.14 A model of the splicing mechanism for removal of a spliceosomal intron. Excision is dependent on snRNPs (U1, U2, etc.). The lariat structure is characteristic of this mechanism. Copyright © 2019 Pearson Education Ltd. All Rights Reserved Animation: Regulation of Gene Expression: Eukaryotes Copyright © 2019 Pearson Education Ltd. All Rights Reserved 36  3/24/2020 13.12 RNA Editing May Modify the Final Transcript Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.12 RNA Editing (1 of 2) RNA editing (‫)التحرير‬ Substitution editing – Identities of individual nucleotide bases are altered; prevalent in mitochondria and chloroplast RNA derived in plants Insertion/deletion editing – Nucleotides are added/deleted from total number of bases – Prevalent in mitochondrial and chloroplast RNAs Copyright © 2019 Pearson Education Ltd. All Rights Reserved 37  3/24/2020 Section 13.12 RNA Editing (2 of 2) Figure 13.15 RNA editing reactions: Deamination of cytidine by the enzyme APOBEC-1 results in uridine (a), whereas deamination of adenosine by the enzyme ADAR produces the noncanonical nucleoetide, inosine (b). Copyright © 2019 Pearson Education Ltd. All Rights Reserved 13.13 Transcription Has Been Visualized by Electron Microscopy Copyright © 2019 Pearson Education Ltd. All Rights Reserved 38  3/24/2020 Section 13.13 Transcription under the Microscope (1 of 2) EM and interpretive drawing of transcription in E. coli – RNA strands emanate (‫ )تنبثق‬from different points along template—numerous transcription events are occurring simultaneously – Cytoplasm ribosomes are not separated physically from chromosome – Polyribosomes are observed in both prokaryotes and eukaryotes Copyright © 2019 Pearson Education Ltd. All Rights Reserved Section 13.13 Transcription under the Microscope (2 of 2) Figure 13.16 Electron micrograph and interpretive drawings of simultaneous transcription and translation of genes in E. coli. As each mRNA transcript is forming, ribosomes attach, initiating translation along each strand. Copyright © 2019 Pearson Education Ltd. All Rights Reserved 39  3/24/2020 Copyright Copyright © 2019 Pearson Education Ltd. 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