Translation of mRNA - Translation of mRNA Translation...

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Unformatted text preview: Translation of mRNA Translation involves the synthesis of cellular proteins from mRNA. enzymes are a particular subset he T of proteins proteins require multiple subunits-have a quaternary s tructure polypeptide has to do with structure, protein has to do with function looking at one gene, one polypeptide....might be an enzyme, might be some other t ype of protein, might be a larger c omplex of some other type of protein Genetic Basis for Protein Synthesis Structural genes encode an amino acid sequence. The RNA that is transcribed from structural genes is called messenger RNA (mRNA). Archibald Garrod proposed that some genes code for the production of a single enzyme. The idea that a relationship exists between genes and protein production was first suggested by Garrod (early 1900s). Garrod studied patients who had the inherited disease alkaptonuria. Patients with this disease accumulate large amounts of homogentisic acid, which causes the urine to appear black. This trait is inherited through a recessive pattern. The disease is due to a defect in phenylalanine metabolism (Figure 13.1). Garrod described this disease as an inborn error of metabolism and suggested that the inheritance of the trait was associated with the production of an enzyme, which in this case was defective. experiments with Neurospora led them to propose the one gene-one can also have one gene-mutliple Beadle and Tatum’s polypeptides enzyme hypothesis. doesn't really need a s upplement problem is you c annot convert the precursor to Ornithine Beadle and Tatum studied nutritional requirements in the bread mold Neurospora crassa, to examine the nature of a gene. They believed that a mutation in a gene that caused a defect in an enzyme needed for synthesis of an essential molecule would prevent the strain from growing on minimal medium. Minimal medium contains a carbon source, inorganic salts, and biotin only. If the medium was supplemented for the one material that the organism could not make due to the mutation, then the organism should be able to grow. Their results (Table 13.1) led them to propose the one-gene-one-enzyme theory. Since then there have been some slight modifications to the theory. Enzymes are only one category of cellular proteins. Some proteins are composed of two or more polypeptides. The term protein is usually used to denote function; polypeptide is used to indicate structure. chances of choosing correct polypeptide by chance alone are 1/20^n n = number of amino acids 1 During translation, the genetic code within mRNA is used to make a polypeptide with a specific amino acid sequence. Translation involves an interpretation of one language into another. The language of mRNA (nucleotides) is translated into the language of proteins (amino acids). This is made possible by the genetic code (Table 13.2). The sequence of bases within the mRNA is read in groups of three, called codons (Figure 13.2). The start codon (AUG) indicates the beginning of a polypeptide sequence. Stop codons (UAA, UAG, and UGA) end translation. The genetic code (Table 13.2) is degenerate, in that more than one codon can specify the same amino acid. These codons are known as synonyms. The third base is usually the degenerate base and is also called the wobble base. The three-base codon was initially studied by Crick (1961), who was studying mutations in bacteriophage T4 (Table 13.4). The start codon (AUG) defines the beginning of the open reading frame (ORF), a sequence of codons that defines the sequence of the polypeptide. Experiment 13A – Synthetic RNA helped to decipher the genetic code. t ake G, and U...make s ome polypeps. 70% G and 30% U. What are chances of getting GGG? = .7x. 7x.7 = .34 put all 20 in but make one radioactive...until y ou cover all 20 of t he amino acids. every tube has 1 RA amino acid and 19 others that are not A number of early researchers were investigating the basis of the genetic code in the early 1960s. One of the key early experiments was done by Nirenberg and Matthaei, who used synthetic RNA molecules to partially decipher the genetic code. Making synthetic mRNA. Polynucleotide phosphorylase in the presence of nucleoside diphosphates will catalyze the covalent linkage of nucleotides to make a polymer of RNA. Because a template is not used, the order of the nucleotides is random. 2 Example using 70% G and 30% U Codon Possibilities GGG GGU GUU UUU UUG UGG UGU GUG The goal Provide information that would help decipher the relationship between base composition in mRNA and particular amino acids. Achieving the goal – see Figure 13.3. 14 were not used at all because they are specifi ed by other combinations using A and C. 6% means the codon appears only %6 of the time glycine must be some c ombination that adds up to 49% because more than one c odon can speciy the same amino acid. c opolymers- repeating units of whatever we started with Percent in the Random Polymer 0.7 X 0.7 X 0.7 = 0.34 = 34% 0.7 X 0.7 X 0.3 = 0.15 = 15% 0.7 X 0.3 X 0.3 = 0.06 = 6% 0.3 X 0.3 X 0.3 = 0.03 = 3% 0.3 X 0.3 X 0.7 = 0.06 = 6% 0.3 X 0.7 X 0.7 = 0.15 = 15% 0.3 X 0.7 X 0.3 = 0.06 = 6% 0.7 X 0.3 X 0.7 = 0.15 = 15% Add the cell-free translation system to each of 20 tubes.. To each tube, add random mRNA polymers of G and U that were made using 70% G and 30% U as shown above. Add a different radiolabeled amino acid to each tube, and add the other 19 nonradiolabeled amino acids. Incubate for 60 minutes to allow translation to occur. Add 15% trichloroacetic acid (TCA), which precipitates polypeptides but not amino acids. Place the precipitate onto a filter and wash to remove unused amino acids. Count the radioactivity on the filter in a scintillation counter. Calculate the amount of radiolabeled amino acids in the precipitated polypeptides. The data and interpreting the data (Figure 13.3) The only radiolabeled amino acids found in the polypeptides were glycine (49%), valine (21%), tryptophan (15%), leucine (6%), cysteine (6%), and phenylalanine (3%). According to the G/U chart shown above, phenylalanine could only be coded for by UUU, leucine and cysteine could be coded for by GUU, UUG, or UGU, etc. This and later experiments established the codons containing only G and/or U to be UUG for leucine, UGU for cysteine, UGG for tryptophan, GUU and GUG for valine, and GGG and GGU for glycine. 3 The use of RNA copolymers and the triplet binding assay also helped to crack the genetic code. Additional work on deciphering the genetic code was performed by Khorana in the 1960s. Khorana used short RNA molecules (2-4 nucleotides) that were linked together to form copolymers (Table 13.5). Because each copolymer usually contained only a few codons, Khorana could determine which codons were responsible for each amino acid. Nirenberg and Leder discovered (1964) that RNA molecules with three nucleotides (triplets) could stimulate ribosomes to bind to tRNA (Figure 13.4). Ribosomes and tRNAs were mixed with a synthesized triplet RNA encoding CCC. Only tRNA carrying proline bound to the ribosome, showing that CCC encodes proline. With additional work, they found that only a specific tRNA would bind to a specific triplet, thus establishing a link between specific codons and specific amino acids. A polypeptide chain has directionality from its amino terminal to its carboxyl terminal end. The polypeptide chain has directionality that parallels the direction of the mRNA chain. During elongation, a peptide bond is formed between the carboxyl group of the last amino acid in the polypeptide chain and the amino group of the amino acid being added (Figure 13.5a). The first amino acid of the polypeptide is said to be at the N-terminal, or amino terminal end. The last amino acid is said to be at the C-terminal, or carboxyl terminal end (Figure 13.5b). Each amino acid contains a different side chain, or R group (Figure 13.6). These have unique chemical properties, which can be combined into general classes. The amino acid sequences of polypeptides determine the structure and function of proteins. Following transcription and translation, the net result is a polypeptide with a defined amino acid sequence. This sequence of amino acids is called the primary structure (Figure 13.7). To become functional, polypeptides must fold into three-dimensional shapes. Secondary structures are formed by regular, repeating shapes. These are either ! helices or " sheets (Figure 13.8a,b). These structures are stabilized by hydrogen bonds. 4 Tertiary structures (Figure 13.8c) occur due to hydrophobic/hydrophilic interactions, ionic interactions, van der Waals interactions, hydrogen bonding, and covalent disulfide bonding. Proteins that contain more than one polypeptide exhibit quaternary structure (Figure 13.8d). Cellular proteins are primarily responsible for the characteristics of living cells and an organism’s traits. The characteristics of a cell depend on the proteins that it makes (Table 13.6). The relationship between genes and traits is illustrated in Figure 13.9. Structure and Function of tRNA The adaptor hypothesis, proposed by Crick and Hoagland, suggested that the position of an amino acid in the polypeptide chain is determined by the bonding between the mRNA and a tRNA containing a specific amino acid. anti codons on t RNA are c omplementary to c odons on mRNA must be a lot of t RNA's to do this because there are a lot of different codons f or same amino acid tRNAs recognize a codon in the mRNA and carry an amino acid that is specific for that codon. The function of a tRNA depends on the specificity between the amino acid it carries and its anticodon. The codons within the mRNA are recognized by tRNAs that carry the correct amino acid to the site of polypeptide synthesis. During the mRNA-tRNA recognition, an anticodon on the tRNA binds to the codon on the mRNA (Figure 13.10). The base sequence of the anticodon complements the codon on the mRNA. The anticodon corresponds to the amino acid that the tRNA is carrying. The cell must produce many different tRNAs to address the needs of the genetic code. Experiment 13B. tRNA functions as the adaptor molecule involved in codon recognition. The experimental test of the adaptor hypothesis was provided by Chapeville (1962). Chapeville’s experiment attempted to determine if the amino acid or the anticodon was important in the recognition of the mRNA codon. This experiment used mRNA that only contained U and G to reduce the number of variables. 5 The hypothesis Codon recognition is dictated only by the tRNA anticodon; the chemical structure of the amino acid attached to the tRNA does not play a role. Testing the hypothesis (Figure 13.11) t here is A tRNA that carries radioactive cysteine, other t RNA are going to pick up other amino acids that are not radioactive when we isolate the cysteine f rom polypeptide, the cysteine s hould be radioactive in test tube - control - all radioactivity should be in c ysteine because cysteine was the tRNA attached to the alanine doesn't matter what the tRNA is carrying, it is still the anticodon... it doesn't matter about the quality of the t RNA... only the quality of the anticodon matters alanine and cysteine will move at different speeds through t he test tube Place cell-free translation system into a test tube. Add amino acids including radiolabeled cysteine (an enzyme within the translation system will attach the radiolabeled cysteine to tRNAcys, and the other tRNAs will have unlabeled amino acids attached to them); incubate and divide into two test tubes. In one test tube, treat the tRNAs with Raney nickel – this removes the –SH group from cysteine, converting it to alanine; do not add Raney nickel to the control tube. Add polyUG mRNA as a template; this mRNA contains codons for cysteine but not for alanine. Allow translation to proceed. Precipitate the newly made polypeptides and isolate on a filter. Hydrolyze the polypeptides to their individual amino acids. Run the sample over a column that separates cysteine and alanine and collect those fractions. Determine the radioactivity in the fractions that would contain cystein and alanine. The data and interpreting the data (Figure 13.11). In the control sample, nearly all of the radioactivity was in the cysteine fraction; this was expected because the only radioactive amino acid attached to tRNAs was cysteine and the mRNA contained cysteine codons. In the Raney nickel-treated sample, a large amount of radioactivity was found in the alanine fraction even though there was no radioactive alanine in the original system and the mRNA did not contain any alanine codons; therefore, the tRNAcys molecules, which carried alanine after the cysteine had been converted to alanine, incorporated alanine into the synthesized polypeptide. These observations indicate that codons in the mRNA are identified directly by tRNA anticodons rather than by the attached amino acids. 6 Common structural features are shared by all tRNAs. All tRNAs share common structural features (Figure 13.12): Three stem loop structures. A few variable sites. makes connection between amino acid and tRNA An acceptor stem with a 3’ single-stranded region. Aminoacyl-tRNA synthetases charge tRNAs by attaching the appropriate amino acid. The enzyme that catalyzes the attachment of amino acids to the 3’ end of the tRNA is the aminoacyl-tRNA synthetase. There are 20 different forms of this enzyme, one for each amino acid. Aminoacyl-tRNA synthetase catalyzes a reaction between an amino acid, a tRNA, and ATP (Figure 13.13). Once the amino acid is attached to the tRNA, it is called a charged tRNA. This process is sometimes called the second genetic code, because there is specificity between the aminoacyl-tRNA synthetase and the tRNA. Mismatches that follow the wobble rule can occur at the third position in codon-anticodon pairing. only a particular t RNA can only fi t into a particular s ynthetase (1 s ynthetase per each amino acid) multiple t RNA fi t into a particular syntetase, c orrect amino acid on c orrect tRNA single tRNA can recognize more than one c odon don't have to make all t RNA to match all codons looking at codon 5' - 3' and anticodon is 3' 5' (matching up tRNA) The genetic code is degenerate. For many amino acid codons, the third base is not crucial to identifying the amino acid. This is called the wobble hypothesis and was first proposed by Crick (1966). When two or more tRNAs can recognize the same codon, they are called isoacceptor tRNAs. This enables a tRNA to recognize multiple codons, thus reducing the total number of different types of tRNAs that a cell must manufacture (Figure 13.14). Ribosome Structure and Assembly The ribosome is the site of translation in both bacterial and eukaryotic cells. Bacterial and eukaryotic ribosomes are assembled from rRNA and proteins. 7 ask maybe about 16s rRNA ---something important about it to process Bacterial cells have a single type of ribosome, while eukaryotic cells have a variety of ribosome types depending on the cellular location. In eukaryotes the most abundant type of ribosome is found in the cytosol. Both mitochondria and chloroplasts contain ribosomes. Ribosomes consist of units called the large and small subunits (Figure 13.15). Each subunit is comprised of proteins and rRNA molecules. The S designation on the subunit refers to the rate that these units sediment when subjected to a centrifugal force. Components of ribosomal subunits form functional sites for translation. interested in what is where and how it is c oming together The shape of the ribosomal subunits is determined by the rRNA. The structure of a bacterial ribosome is illustrated in Figure 13.16c. Ribosomes contain discrete sites. These are the peptidyl (P) site, aminoacyl (A) site, and exit (E) site. Stages of Translation The three stages of translation are initiation, elongation, and termination (Figure 13.17). The initiation stage involves the binding of mRNA and the initiator tRNA to the ribosomal subunits. Initiation involves the binding of an mRNA, tRNA, and the ribosomal subunits (Figure 13.18). In bacteria the initiator tRNA carries a methionine that has been modified to a Nformylmethionine. The steps of initiation in bacteria include (Figure 13.18): Initiation factor IF3 promotes the binding of the mRNA to the 30S subunit. A ribosomal binding site, also called the Shine-Dalgarno sequence (Figure 13.19), in the mRNA complements a short region of the 16S rRNA. IF2 promotes the binding of the initiator tRNA, which contains N-formylmethionine. IF2 and IF3 are released, allowing the association of the 50S subunit, forming a 70S initiation complex. 8 instead of Delgamo In eukaryotes, the processes are similar, except: The initiator tRNA contains methionine (not modified). The process involves a larger number of initiation factors (Table 13.7). The 7-methylguanosine cap at the 5’ end of the mRNA is active in the initial binding of the mRNA to the ribosome. The ribosome begins at the 5’ end of the mRNA and scans for an AUG start site, which is not always the first start site. Polypeptide synthesis occurs during the elongation stage. Elongation involves adding amino acids, one at a time, to the polypeptide chain. In eukaryotes the rate of elongation is 6 amino acids per second. In bacteria the rate of elongation is 15-18 amino acids per second. The process of elongation is illustrated in Figure 13.20 and involves the following general steps: A charged tRNA, determined by the mRNA codon, binds to the A site. The enzyme peptidytransferase catalyzes bond formation between the polypeptide chain and the amino acid in the A site. The ribosome translocates itself (i.e., the ribosome moves) one codon toward the 3’ end of the mRNA. The uncharged tRNA is released from the E site. The process is repeated until a stop codon is reached. The 16S rRNA can determine if an incorrect tRNA is located in the A site and will prevent elongation until the tRNA is removed. This is called a decoding function, and it accounts for the high fidelity of translation. Termination occurs when a stop codon is reached in the mRNA. Stop codons, also called nonsense codons, are not recognized by a tRNA but are recognized by release factors (RF). In bacteria there is a specific release factor for each stop codon. In eukaryotes there is a single release factor. 9 in prok - all occuring inThe c ytoplasm. can couple t ranscirption and t ranslation in euk - trans in nucleus and translation in c ytoplasm following events occur during termination (Figure 13.21): The release factor binds to the A site. The polypeptide is released from the P site, followed by the tRNA. The ribosomal subunits and mRNA dissociate. Bacterial translation can begin before transcription is completed. B can occur in endoplasmic ecause bacteria do not possess a nucleus, may occur simultaneously (Figure 13.22). reticulum the processes of transcription and translation An mRNA transcript that has multiple ribosomes attached to it is called a polyribosome or polysome. The amino acid sequence of proteins contains sorting signals Sorting signals direct proteins to their correct locations within a cell. In eukaryotes sorting can occur during translation (cotranslational) or following translation (posttranslational). This is illustrated in Figure 13.23. Cotranslational import occurs when protein synthesis begins in the cytosol, but then moves to the endoplasmic reticulum for completion. This is typically associated with proteins that are bound for the plasma membrane, endoplasmic reticulum, Golgi, or the lysosomes. Short amino acid sequences, called sorting signals, direct each protein to its correct location (Table 13.8). Please see the Conceptual Summary and Experimental Summary for Chapter 13 on pages 354 and 355. This lecture outline was prepared from Genetics: Analysis and Principles, by Brooker, 2009 (3rd edition). It contains phrases and entire sentences taken verbatim from that source, and is in no way meant to represent original work by Mark Bierner. 10 ...
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This note was uploaded on 05/19/2011 for the course BIO 325 taught by Professor Saxena during the Spring '08 term at University of Texas at Austin.

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