11bBIS1012012TranslatLect11b

11bBIS1012012TranslatLect11b - BIS101-001: Genes and Gene...

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Unformatted text preview: BIS101-001: Genes and Gene Expression Translation, Protein Synthesis and the Genetic Code Lecture #11b Chapter 9 March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 1 In this lecture Translation: The process by which genetic information encoded in a four base alphabet - A,G,C and U is converted into protein which uses an alphabet consisting of 20 amino acids. Genetic Code: The genetic code is a degenerate, commaless, non-overlapping triplet code. The "coding sequence" of a gene is defined by the open reading frame (ORF). Shine Dalgarno (SD) sites align mRNA on the ribosome. Translation: Synthesizing proteins from mRNAs. Three stages: initiation, elongation, and termination. Adapter Molecules: The lack of affinity between nucleotides and amino acids makes it necessary for the cell to enlist the help of two "adapter molecules" transfer RNAs (tRNAs) and aminoacyl synthetases (AAS). Wobble Hypothesis explains how there are more than 20 but less ant 61 tRNAs for translation. Chemical modifications of tRNAs make to recognizable by initiation factors and the second adaptor molecule, aminoacyl synthetase. Release factors recognize "stop codons" to terminate translation elongation. BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 2 March 19, 2012 Translation Genetic Code and Protein Synthesis March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 3 Introduction One of the greatest challenges facing molecular biologists in the 50's and 60's was determining the mechanism by which the "two great polymer languages" (i.e., nucleic acids and proteins) communicated with each other. This was an especially difficult problem because: q q (1) free nucleotides and amino acids have no strong affinity for each other, (2) although there are only 20 amino acids (the building blocks of proteins), the genetic code consists of 64 codons. This makes the genetic code degenerate. For these reasons, translating the genetic code into protein is a complex process, requiring several components and the action of two adapter molecules: tRNA and the enzyme aminoacyl synthase. BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 4 March 19, 2012 Initiation and Elongation of Translation http://www.youtube.com/watch?NR=1&feature=fvwp&v=Ikq9AcBcohA http://www.youtube.com/watch?v=5bLEDd-PSTQ March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 5 Translation Links Nucleotides and Amino Acids X DNA mRNA tRNA Aminoacyl Synthetase Amino Acids X March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 6 Requirements for Translation mRNA (codons) Ribosomes Transfer RNA (tRNA) Aminoacyl synthetase Amino acids Initiation, elongation and termination factors ATP, GTP March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 7 Requirements for Translation mRNA (codons) Ribosomes Transfer RNA (tRNA) Aminoacyl synthetase Amino acids Initiation, elongation and termination factors ATP, GTP March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 8 Genetic Code Table March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 9 Open Reading Frame (ORF) 5'-THEFATCATATETHEBIGRAT-3' 5'-THE FAT CAT ATE THE BIG RAT-3' 5'-T HEF ATC ATA TET HEB IGR AT-3' Frameshifts +1 0 5'-TH EFA TCA TAT ETH EBI GRA T-3' 0+1+2 5'A T G C C T A T T G G G A T C T A T T A A T C C C3' 3'T A C G G A T A A C C C T A G A T A A T T A G G G5' 2+1+0 6 possible ORFs for any segment of DNA March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 10 +2 DNA can be read in 6 possible ways The two strands of DNA can be read in the 5' 3' direction, and on either strand there are three possible start point, at the 1st, 2nd, or 3rd nucleotides. Each of these possibilities is called a reading frame: an Open Reading Frame (ORF) is a long stretch of DNA that can be read without encountering a stop codon. ORFs are considered candidate for protein-coding exon regions: the inferred amino-acid sequence can be compared with GenBank to identify possible analogous proteins. Because 3 of the 64 possible codons are stop codons, a stop codon should occur at random approximately every 20 codons : the occurence of multiple stop codons in a particular reading frame indicates that it does not code for a polypeptide. Ordinarily, only one reading frame is open. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 11 The Genetic Code is a Degenerate Code As predicted by the physicist George Gamov, a genetic code consisting of only two letter words would provide only 16 words, four short of the total number of amino acids. With a code consisting of three letters, (64 codons) there would be more than one codon for each amino acid. This means that the genetic code is degenerate. Amino Acid Leu, Ser, Arg Gly, Pro, Ala, Val, Thr Ile Phe, Tyr, Cys, His, Glu Gln, Asn, Asp, Lys Met, Trp Nonsense (ochre) Nonsense (opal) Nonsense (amber) # Synonymous Codons 6 4 3 2 0 Total Codons 18 20 3 18 2 UAA UGA UAG Total 64 March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 12 Requirements for Translation mRNA (codons) Ribosomes Transfer RNA (tRNA) Aminoacyl synthetase Amino acids Initiation, elongation and termination factors ATP, GTP March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 13 Requirements for Translation: Ribosomes Ribosomes: The prokaryotic ribosome consists of 2 subunits, 30S and 50S, to make a 70S structure. The bacterial ribosome consists of 3 ribosomal RNA (5S and 23S in the 50S subunit: 16S in the 30 subunit) and 52 proteins (31 in the 50S and 21 in the 30S subunit). The ribosome is the focal point in the cell on which the many steps of translation take place. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 14 Sucrose Gradients and "S" Values Macromolecular sedimentation coefficients (s) typically range from 10-11 to 10-13 seconds. In honor of Dr. Svedberg, the sedimentation coefficient was changed to the Svedberg unit, or "S" value. For most macromolecules, S values range from 1 (10-11) to 100 (10-13). A typical velocity sedimentation ultracentrifugation experiment involves the use of a 5-20% sucrose gradient prepared in the centrifuge tube (see Figure below. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 15 Eukaryotic Ribosome The eukaryotic ribosome is similar to prokaryotic ribosomes except for is size which is reflected in the number proteins and size of the ribosomal RNAs. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 16 3-D Structure of Bacterial Ribosome 30S subunit 50S subunit 70S subunit March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 17 Requirements for Translation mRNA (codons) Ribosomes Transfer RNA (tRNA) Aminoacyl synthetase Amino acids Initiation, elongation and termination factors ATP, GTP March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 18 Transfer RNA structure tRNA Transfer RNAs are 70-90 bases long and provide the first of two links between mRNA and amino acids. The 3' ACC end is common to all tRNAs and forms a bond with the proper (cognate) amino acid and the three bases in the anticodon loop hydrogen bonded with a complementary codon in the mRNA. There must be at least as many different tRNAs as there are amino acids but in reality, there are more. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 19 L-Form Structure of tRNA Intramolecular complementarity (like shRNA), enables tRNA to adopt a complex, double helical structure. The 3-D structure of tRNA indicate that it is not a cloverleaf structure but rather an upside down "L" structure with stress and unusual hydrogen bonds. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 20 L-Form Structure of tRNA March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 21 Modified Bases in tRNA The nitrogenous bases in tRNAs are highly modified. This gives each tRNA a unique "signature." These unique signatures permit each tRNA to recognize (or be recognized by) its cognate (corresponding) aminoacyl synthetase enzymes. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 22 Modifications of Specific tRNAs March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 23 Two Methionine tRNAs All prokaryotic and eukaryotic translation begins with methionine, although this amino acid may be removed after translation terminates. In prokaryotes, translation initiates with a specially modified methionine called "formyl-methionine." This special methionine is required for the formation of the initiation complex. Since methionine residues frequently occur with the mRNA, how does the cell prevent the incorpation of f-met within the polypetide? Special structural modifications on the tRNAf-met is recognized by initiation factor 2 (IF2) and by methionyl transformylase, the enzyme responsible for formylating methionine. Since the special tRNAf-met can not bind the ribosome without the help of IF2, f-met is not incorporated at other AUG codons on the mRNA. Likewise, normal tRNAmet can not be used to initiate translation because it is not recognized by IF2 and methionyl transformylase. However, the Met codon, AUG, often appears several times in mRNA. 24 March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 4 Differences between f-met tRNa met-tRNA* The following modifications permit the fMet-tRNA to be recognized by IF2 q (1) an unbound C-A base pair at the 5' end of the tRNA; q (2) lack of an alkylated adenosine in the anticodon loop; q (3) the presence an A in the TC loop instead of a G and q (4) C-G bonding in the stem above the anticodon loop. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 25 Wobble Hypothesis and Wobble Rules Because there are 61 codons for 20 amino acids, one could reason that there must be 61 tRNA. In reality, there are fewer tRNAs than codons. This means that one tRNA must possess the ability to recognize more than one codon. This explanation was first proposed by Francis Crick as the Wobble Hypothesis, which states that because of the unusual physico-chemical environment in the anticodon loop of the tRNA, the 3rd base toward the 5' side of the loop can temporarily form hydrogen bonds with other bases. This enables one tRNA to interact with synonymous codons that differ at the third position. (e.g. UUU and UUC = phe). According to the Wobble (base pairing) Rules, the 3rd base toward the 5' end of the anticodon loop can hydrogen bond with certain bases in the 3rd position of the codon: BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 26 March 19, 2012 Wobble in the Anticodon Loop March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 27 Wobble Base Pairing Rules Base at 5' end of Anticodon I (deaminated A) G U C A Base at 3' end of Codon U, A, C C, U A, G G U March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 28 Isoaccepting tRNA Using the six codons for serine, we can demonstrate how the wobble rule can reduce the number of tRNAs necessary to recognize synonymous codons: 1 5'UCU 3'AGA 3'AGI 2 UCC AGG 3 UCA AGU 4 UCG AGC 5 AGC UCG 6 AGU UCA Codon # Codon Anticodon Using the Wobble Rules, a minimum of 32 anticodons can accommodate the 61 codons. tRNAs that recognize more than one codon are called isoaccepting tRNAs. BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 29 March 19, 2012 Requirements for Translation mRNA (codons) Ribosomes Transfer RNA (tRNA) Aminoacyl synthetase Amino acids Initiation, elongation and termination factors ATP, GTP March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 30 Charging the tRNA with Aminoacyl Synthetase* Purves et al, 1997, Life the Science of Biology W.H. Freeman March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 31 Requirements for Translation mRNA (codons) Ribosomes Transfer RNA (tRNA) Aminoacyl synthetase Amino acids Initiation, elongation and termination factors ATP, GTP March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 32 20 Amino Acids and Their Properties March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 33 Requirements for Translation mRNA (codons) Ribosomes Transfer RNA (tRNA) Aminoacyl synthetase Amino acids Initiation, elongation and termination factors ATP, GTP March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 34 Binding of mRNA to the 30S ribosomal subunit Near the end of the promoter sequence are two additional sequences that play important roles in the initiation of translation. These are the SD site (Shine Dalgarno) and methionine codon, ATG (or AUG in the mRNA). The SD site, also referred to as the ribosome binding site (RBS), is required for aligning the mRNA on the 30S subunit of the ribosome. This is accomplished by hydrogen bonding between the SD site on the mRNA and complementary sequences at the 3' end of the 16S rRNA in the 30S subunit. Once aligned, the methionine codon (AUG) is placed such that when bound by the aminoacyl methionine-charged tRNA, the methionine will be over the P site on the 50S subunit. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 35 Shine Dalgarno Sites in Prokaryotic mRNAs These SD sites in prokaryotes are conserved. Shown below are 8 SD sites that have been used to generate the consensus sequence 5'-AGGAG-3' March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 36 Bonding of the SD site to the 3' end of 16S rRNA Shown below is hydrogen bonding between the 3'-end of the 16S rRNA (UCCUCCA) to the 5'-end of the (AGGAGGU) of the mRNA. This binding is part of the "pre-initiation" complex. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 37 Three Stages of Translation Initiation: In prokaryotes, protein synthesis begins with Nformyl-methionine (F-met)-tRNA bound to the 30S subunit in a preinitiation complex. The tRNAf-met is positioned using the ribosome binding site, so that the F-met amino acid will fit into the peptidyl site (P-site) of the large (50S) subunit. Elongation: The mRNA is read on the ribosome in the 5' to 3' direction. The amino acid in the P site of the large subunit (50S) is transferred to the amino acid in the A site on the same subunit through the formation of a peptide bond. This complex is then transferred back to the P site. Termination: The codons, UAA, UAG and UGA (nonsense codons), together with releasing factors, terminate protein synthesis. Nonsense or stop codons do not bond to tRNAs, but rather they are bound by release factors RF1 and RF2. RF1 recognizes the UAA and UAG codon while the RF2 recognized the UAA and UGA codons. BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 38 March 19, 2012 Translation Initiation: The Pre-initiation Complex* Translation initiates with the formation of a preinitiation complex consisting of the messenger RNA, a 30S ribosomal subunit and a special aminoacyl-tRNA molecule, tRNAf-met. Bacterial protein synthesis initiates with Nformyl methionine at a methionine codon, (AUG) near the 5' end of the mRNA. The anticodon of tRNAf-met base pairs with the initiation codon on the mRNA. Initiation factors 1 and 3 (IF1 and IF3) bind to the 30S subunit and blocking the binding of the 50S subunit. IF2 binds to tRNAf-met and brings it to the 30S subunit. Once the tRNAf-met is bound to the 30S subunit, IF2 hydrolyzes GTP, causing the dissociation of IF1, IF2, and IF3. The 50S subunit contains two sites, a P (peptidyl) site and an A (aminoacyl) site. In the 70S initiation complex the tRNAf-met lies in the peptidyl site. BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 39 March 19, 2012 Peptide Bond Formation March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 40 Summary of Gene Expression Components Not shown at the right are the many "regulatory DNA sequences" (e.g., promoters, terminators, enhancers, ribosome binding sites) that are required by specific expression of prokaryotic and eukaryotic genes. Also not shown are all the transcription factors required for eukaryotic transcription. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 41 Summary Translation: The process by which genetic information encoded in a four base alphabet - A,G,C and U is converted into protein which uses an alphabet consisting of 20 amino acids. Genetic Code: The genetic code is a degenerate, commaless, non-overlapping triplet code. The "coding sequence" of a gene is defined by the open reading frame (ORF). Shine Dalgarno (SD) sites align mRNA on the ribosome. Translation: Synthesizing proteins from mRNAs. Three stages: initiation, elongation, and termination. Adapter Molecules: The lack of affinity between nucleotides and amino acids makes it necessary for the cell to enlist the help of two "adapter molecules" transfer RNAs (tRNAs) and aminoacyl synthetases (AAS). Wobble Hypothesis explains how there are more than 20 but less ant 61 tRNAs for translation. Chemical modifications of tRNAs make to recognizable by initiation factors and the second adaptor molecule, aminoacyl synthetase. Release factors recognize "stop codons" to terminate translation elongation. BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 42 March 19, 2012 ...
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This note was uploaded on 03/18/2012 for the course BIS 101 taught by Professor Simonchan during the Winter '08 term at UC Davis.

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