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Chapter 16

Chapter 16 Transcription and Translation.docx

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Department
Biology
Course
BIO152H5
Professor
Fiona Rawle
Semester
Fall

Description
Chapter 16 Transcription and Translation Key Concepts: - After RNApolymerase binds DNAwith the help of other proteins, it catalyzes the production of an RNAmolecule whose base sequence is complementary to the base sequence of the DNAtemplate strand. - Some sections of an RNAare enclosed by gene regions called exons, while others are cncoded by gene regions called introns. During RNAprocessing, introns are removed and the ends of the RNAreceive a cap and tail. - Inside ribosomes, mRNAs are translated to proteins via intermediary molecules called transfer RNAs. Transfer RNAs carry an amino acid and have a three-base-pair anticodon, which binds to a three-base-long mRNA codon. The amino acid carried by the transfer RNAis then added to the growing protein via formation of a peptide bond. - Mutations are random changes in DNAthat may or may not produce changes in the phenotype. 16.1 Transcription in Bacteria -RNApolymerase performs a template-directed synthesis in 5’’direction. Does not require primer to begin transcription. -Transcription occurs when RNApolymerase matches the base in a ribonucleotide triphosphates with the complementrary base in a gene. -RNApolymerase catalyzes the formation of a phosphodiester bond between the 3’end of the growing mRNAchain and the new ribonucleotide. -Only one of the two DNAstrands is used as a template by RNApolymerase. Other strand us non-template or coding strand. Coding strand has similar sequence to RNA. RNAPolymerase Structure and Function -X-ray crystallography -Large, globular, several prominent channels running through interior. -Enzyme’s active site where phosphodiester bonds form is located where several channels intersect. -DNAfits into channel, double helix separates, expose single strand at active site. Initiation: How Does Transcription Begin? -Detachable protein subunit “sigma” must bind to RNApolymerase for transcription to begin. -Holoenzyme: RNApolymerase and sigma. Consists of core enzyme (RNA polymerase), which contains active site for catalysis, and other required proteins. -Holoenzyme binds to promoters (binding sites on DNAwhere transcription begins). Sigma guides RNApolymerase to specific locations where transcription begins. - -10 box: TATAAT. Centered 10 bases from the point where RNApolymerase starts transcription. 10 bases upstream from transcription start site. -Downstream: DNAlocated in direction RNApolymerase moves during transcription. -Upstream: DNAlocated in the opposite direction. - +1 site: where transcription begins. - -35 box: TTGACA occurs in promoters. 35 bases upstream from +1 site. - Transcription begins when sigma binds to -35 and -10 boxes. Sigma, not RNApolymerase, makes initial contact when DNAstarts transcription in bacteria. Sigma tells RNA where and when to start synthesizing RNA. -Sigma binds, DNAunwinds creates 2 single strands. Template threaded through channel that leads to active site inside polymerase. NTPs enter channel at bottom of enzyme and diffuse to active site. -RNA polymerization begins when NTP pairs with complementary base on template strand. Reaction is exergonic and spontaneous because NTPs have so much potential energy because of phosphates. Sigma released when RNAsynthesis is under way. Elongation and Termination Elongation: -RNApolymerase moves along DNAtemplate in 3’5’direction synthesizing RNA. -In the interior of the enzyme, enzyme’s zipper helps open double helix at upstream end. Rudder helps steer the template and non-template strands through channels inside the enzyme -Active site catalyses addition of nucleotides to the 3’end. -DNAgoes into and out of one group in enzyme, ribonucleoside triphosphates enter another, growing RNAexits to the rear. Termination: -Stops when RNApolymerase reaches a stretch of DNAthat functions as transcription termination signal. -Leads to formation of hairpin in mRNA, disrupting the transcription complex. 16.2 Transcription and RNAProcessing in Eukaryotes -Proteins called basal transcription factors initiate eukaryotic transcription by matching the enzyme with the appropriate promoter region in DNA. -In bacteria a single sigma protein binds to a promoter and initiates transcription, but in eukaryotes many basal transcription factors are required to initiate transcription. In eukaryotes, the machinery required to start transcription is complex. -Many of the eukaryotic promoters required by RNApol II include a unique sequence called the TATAbox, located 30 base pairs upstream of the transcription start site. Some of the promoters recognized by pol II do not contain a TATAbox, however. In addition, RNApol I and pol III interact with entirely different promoters. -In eukaryotes transcription is followed by several important RNAprocessing steps that result in the production of an mRNAthat leaves the nucleus. Name of enzyme Type of Gene Transcribed RNA pol I Genes that code for most of the large RNA molecules (rRNAs) found in ribosomes. RNA pol II Protein-coding genes (produce mRNAs) RNA pol III Genes that code for transfer RNAs and genes that code for one of the small RNAmolecules (rRNAs) found in ribosomes. RNA pol II and III RNAmolecules found in snRNPs The Startling Discovery of Eukaryotic Genes in Pieces -Introns are sections of genes not expressed in final mRNAproduct. Exons are expressed. Eukaryotic genes are much larger than their corresponding mature RNAtranscripts. Exons, Introns, and RNAsplicing Primary RNAtranscript: Transcription of eukaryotic genes by RNApolymerase creates this, contains both exon and intron regions. -As transcription proceeds, introns are removed by splicing. -Splicing is catalyzed by a complex of proteins and small RNAs known as small nuclear ribonucleoproteins or snRNPs (snurps). -snRNP binds to 5’exon-intron boundary. Other snRNPs arrive to form a multipart complex called spliceosome. -Intron forms loop with adenine at its base.Adenine cuts loop out. Phosphodiester bond links exons on either side. -Both cutting and rejoining reactions are catalyzed by RNAmolecules in the spliceosome. Adding Caps and Tails to Transcripts -5’end of eukaryotic RNAemerges from RNApolymerase, enzymes add 5’cap (consisting of 7- methylguanylate and 3 phosphate groups) -Enzyme cleaves 3’end of most RNAs after transcription, another enzyme adds a tract of 100- 250 As (poly(A)tail) -With the addition of the cap and tail, processing of primary RNAtranscript is complete. Product is mRNA. Transcription in Bacteria and Eukaryotes Aspect Bacteria Eukaryotes RNApolymerase One Three; each produces a different class of RNA. Promoter structure Typically contains a -35 box and Complex and variable; often include a -10 box. TATAbox -30 from start of gene. Protein(s) involved in Sigma; different versions of Many basal transcription factors contacting promoter sigma bind to different promoters RNAprocessing steps None; translation occurs while Extensive; several processing steps occur transcription is still under way in nucleus before RNAis exported to cytoplasm for translation: 1. Enzyme-catalyzed addition of 5’cap. 2. Splicing (intron removal) by spliceosome. 3. Enzyme-catalyzed addition of 3’poly(A) tail. 16.3 An Introduction to Translation -ribosomes are the site of protein synthesis. -Britten pulse-chase experiment- fed radioactive sulfate to E.coli. Radioactive atoms were found in free amino acids or ribosomes. Later, all were found on completed proteins. -Crick suggested that ome sort of adapter molecule holds amino acids in place while interacting directly and specifically with a codon in mRNAvia hydrogen bonding. 16.4 The Role of Transfer RNA Aminoacyl tRNA: AtRNAmolecule that gets covalently linked to amino acid. Aminoacyl tRNAsynthetases: Catalyze addition of amino acids to tRNA. How AminoAcids are Loaded onto tRNAs What happens to amino acids attached to tRNAs? 1. Active site on aminoacyl tRNA Hypothesis: Aminoacyl tRNAs transfer amino acid to growing synthetase bindsATP and amino acid. polypeptides. Each aminoacyl tRNAsynthetase is
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