Prokaryotes polypeptide chain initiation

Synthesis of all polypeptide chains In prokaryotic and eukaryotic cells begins with the amino acid methionine. In most mRNAs, the start (initiator) codon specifying this amino-terminal methionine is AUG. In a few bacterial mRNAs, GUG is used as the initiator codon, and CUG occasionally is used as an initiator codon for methionine in eukaryotes. The three codons UAA, UGA, and UAG do not specify amino acids but constitute stop (termination) codons that mark the carboxyl terminus of polypeptide chains in almost all cells. The sequence of codons that runs from a specific... 

The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5 -end to its 3 -end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNAs often have several coding regions, that is, they are polycistronic (see p. 420). Each coding region has its own initiation codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA codes for only one polypeptide chain, that is, it is monocistronic. The process of translation is divided into three separate steps initiation, elongation, and termination. The polypeptide chains produced may be modified by posttranslational modification. Eukaryotic protein synthesis resembles that of prokaryotes in most details. [Note Individual differences are mentioned in the text.]... 

In addition, several ribosomes can independently and simultaneously translate a mRNA molecule and, hence, synthesize several identical polypeptide chains concurrently (Figure 12.3). Such clusters or groups of ribosomes are called polyribosomes or polysomes. The number of attached ribosomes depends on the size of the mRNA and how frequently ribosomes can initiate at the start of a gene sequence. Because RNA transcription and translation are neither temporally nor spatially separated in prokaryotes, it is possible for translation to begin before transcription is completed. However, we have already noted that prokaryotic mRNAs have short half-lives this is probably a result of their continuous degra-... 

The codon AUG has two functions. It corresponds to the amino acid methionine when AUG occurs within a coding sequence in the mRNA, i.e., within a polypeptide chain. It also serves as a signal to initiate polypeptide synthesis—with methionine for eukaryotic cells but with N-formylmethionine for prokaryotic cells. How the protein-synthesizing system distinguishes an initiating AUG from an internal AUG is discussed below. The codon GUG also has both functions, but it is only rarely used in initiation. Once initiation has occurred at an AUG codon, the reading frame is established and the subsequent codons are translated in order. 

Eukaryotic initiator tRNA molecules differ from the prokaryotic initiator molecule in several ways. The most striking difference is that whereas eukaryotic organisms produce both a normal tRNA and an initiator tRNA, which is also charged with methionine, the methionine does not undergo formylation. In eukaryotes, the first amino acid in a growing polypeptide chain is Met and not fMet. The codon for both kinds of tRNA molecules in eukaryotes is AUG, just as for prokaryotes. 

The methionine codon AUG functions both to initiate a polypeptide chain and to direct methionine incorporation into internal positions in a protein. By what mechanisms are the AUG start codons selected in prokaryotes ... 

The first amino acid that starts the process of protein synthesis in prokaryotic (bacterial) cells is a derivative of methionine. This compound, A -formylmethionine, initiates the growing polypeptide chain as the N-tenninal amino acid. The fact that most proteins do not have Af-formylmethionine as the N-terminal amino acid indicates that when protein synthesis is completed, the iV-formylmethionine is cleaved from the finished protein. 

P-site. The peptidyl site contains the tRNA bound to the polypeptide chain. NB The initiating met-tRNA (or formylmet-tRNA in prokaryotes) binds to this site to start assembly of the ribosome and then polypeptide synthesis. 

Diagrammatic representation of translation on prokaryotic ribosomes. The elongation cycle starts by interaction of the 70S initiation complex with fMet- tRNA EFTu GTP. In all subsequent rounds of the cycle, fMet-tRNArEFT tGTP interacts with the mRNA ribosome complex carrying the growing polypeptide chain. Termination occurs when n amino acids have been incorporated, where n represents the number of codons between the initiation codon AUG and the termination codon (in this example UAA). 

The information contained in the base sequence of the mRNA template is interpreted in sequences of three bases called codons each codon represents one amino acid. Therefore, the unit of information is the codon. Since there are four major bases in mRNA, 4 (i.e. 64) different codons are possible. The 64 triplets constitute the genetic code (Table 17.1). All codons have been assigned to amino acids or punctuation signals. Three triplets (UAA, UAG and UGA) are not complemented by anticodons on tRNAs and serve to signal that the polypeptide chain has been completed. Of the other 61 triplets which have complementary tRNAs, two (AUG and GUG) have additional roles in the initiation of protein synthesis. Since there are only 20 amino acids, most amino acids are specified by more than one codon, i.e. the code is degenerate. The genetic code applies to prokaryotes and eukaryotic nuclear and chloroplast mRNAs but not to... 

This claim is based on the following observations (1) Like that of their prokaryotic ancestors, mitochondrial translation uses fMet-tRNA (rather than the Met-tRNA used by cytoribosomes) as the initiator. (2) It can do so successfully because the transformylase which converts the Met-tRNA into its fMet derivative is mitochondrial in its localization. (3) The formation of the initiation complex can be monitored by the transfer of labeled formate (f ) to f Met to puromycin, resulting in its quantitative conversion to f Met-puro —a reaction that is restricted both in vitro and in vivo to the mitochondrial fraction and can be shown to go on with linear kinetics for extended periods. (4) Retention of f Met on nascent polypeptide chains is restricted to mitochondrial polyribosomes and can be used as a specific means for the identification and characterization of the latter. (5) Mitochondria, at least of the yeast species examined by us, appear to be deficient in both a deformylase capable of removing formate from fMet, whether free or on polypeptides, as well as in peptidases capable of removing either this component itself or small peptides from the N-terminal end. (6) Initiation by fMet is absent in p" mutants. In principle then, presence of formate as N-terminal fMet in a polypeptide provides an unambiguous means for its identification as having been synthesized on mitoribosomes. In practice, although feasible, as will be shown in the next section, this is difficult because it requires the prior... 

Every polypeptide has an amino terminus and a carboxyl terminus. In both prokaryotes and eukaryotes, synthesis begins at the amino terminus. For a protein having the sequence HaN-Met-Trp-Asp... Pro-Val-COOH, the f Met (or Met) is the initiating amino acid and Val is the last amino acid added to the chain. Translation of mRNA molecules occurs in the 5 3 direction. 

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