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Mechanism of Protein Biosynthesis Perer LENGYEL a DIETER SOIL Department Ofmolecular Biophysics, Yale University, New Haven, Connecticut 06520

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Mechanism of Protein Biosynthesis Perer LENGYEL a DIETER SOIL Department Ofmolecular Biophysics, Yale University, New Haven, Connecticut 06520 BACTRIOLOGICAL REviEws, June 1969, p. 264-301 Vol. 33, No. 2 Copyright @ 1969 American Society for Microbiology Printed In US.A. Mechanism of Protein Biosynthesis PErER LENGYEL A DIETER SOIL Department ofMolecular Biophysics, Yale University, New Haven, Connecticut 06520 INTRODUCTION ......................................................... 265 Abbreviations ......................................................... 265 Cell-Free Protein Synthesizing Systems........................................ 265 AA-tRNA SYNTHETASES .................................................... 266 Isolation and Properties of AA-tRNA Synthetases................................ 266 Reaction Mechanism......................................................... 267 Complexes Between AA-tRNA Synthetases and tRNA ............................ 268 Genetics of Synthetases ...................................................... 268 TRANSFER RNA......................................................... 268 Sequence oftRNA.........................................................tN 268 Genetics of tRNA......................................................... 270 Recognition of tRNA by the AA-tRNA Synthetase............................... 270 Minor and Rendant tRNA Species ........................................... 270 Virus Infection and tRNA.................................................... 271 Inactivation of host tRNA ................................................... 271 Phage-coded tRNA........................................................ 271 Cytokinins and tRNA....................................... ..................271 Cell Wall Synthesis and tRNA ................................................ 272 Differentiated Cells and tRNA................................................ 272 Other tRNA Reactions ....................................................... 273 RIBOSOMES ................ 273 Ribosomal Proteins of the 30S Subnit. 273 Ribosomal RNA ......................................................... 273 SS RNA.......................................................... 273 16S and 23S RNA......................................................... 273 Reconstitution of Active Ribosomes from RNA and Protein........................ 274 PEPTIDE CHAIN INITIATION............................................... 274 Initiator of Peptide Chains: fMet-tRNA ............ ............................ 274 Fate of the Formyl and the fMet Residues....................................... 275 Coding Specificity and Function of tRNAF and tRNAm........................... 275 Phasing Activity of Initiator Codons........................................... 275 Translation of Polygenic mRNA............................................... 276 Initiation Factors......................................................... 276 Exchange of Ribosomal Subunits During Protein Synthesis ........................ 277 Role of 30S Subunits in Imtiation .................. ............................ 277 Process of Initiaon......................................................... 277 Steps in tation......................................................... 277 Characteristics of initiation complexes ........................................ 277 Puromycin and the tRNA binding sites of the ribosome.......................... 279 Site of binding of fMet-tRNAr to ribosomes ................................... 279 Role of initiation factors.279 Role of the formyl residue and of tRNAFi; in itiatiOn.280 Possible Involvement of fMet-tRNAF in the Regulation of RNA Synthesis........... 280 Peptide Chain Initiation Various Organisms ................................... 281 Procaryotic cells ......................................................... 281 Eucaryotic cells......................................................... 281 PEPTlDE CHAIN ELONGATION............................................ 281 Elongation Factors......................................................... 281 Process of Elongation........................................................ 282 Outline of the steps in elongation ............................................ 282 AA-tRNA Binding......................................................... 283 Peptide Bond Formation...................................................... 284 Translocation......................................................... 285 Problems Arising from the Dual Specificities of the AUG and GUG Codons ........ 286 PEPTIDE CHAIN TERMINATION........................................... 287 Termination Signals ......................................................... 287 Release Factors and the Mechanism of Termination .............................. 287 Fate of the mRNA-Ribosome Complex After Chain Termination ................... 288 264 VOL. 33, 1969 MECHANISM OF PROTEIN BIOSYNTHESIS 265 PROBLEMS AND CONCLUSIONS.......................... 289 AA-tRNA synthetases............................ 289 Transfer RNA.......................... 289 Ribosomes..... 289 Peptide chain initiation .................................................... 289 Peptide chain elongation.................................................... 290 Peptide chain termination ................................................... 290 LITERATURE CITED........................................................ 291 INTRODUCTION charged tRNA species, for example, Ala- The amino acid sequence of a particular protein tRNACYs; different tRNA species capable of is specified by the sequence of nucleotides in a accepting the same amino acid, isoaccepting particular segment of the deoxyribonucleic acid tRNAs; N-acetyl AA-tRNA, acAA-tRNA (e.g., (DNA). The process of protein synthesis consists acPhe-tRNA); phe-phe-tRNA, diphe-tRNA; phe- of two stages. First, the DNA is transcribed into phe-phe-tRNA, triphe-tRNA; aminoacyl oligo- a ribonucleic acid (RNA) intermediate, messenger nucleotides derived from AA-tRNA, for example, RNA (mRNA), which has a ribonucleotide se- CpA-Gly or CACCA-acLeu; AA-tRNA synthe- quence complementary to that of the deoxyribo- tase aminoacyladenylate complex, E-AA-AMP; nucleotide sequence of one of the strands of the mRNA which is translated into more that one DNA serving as template (transcription) (120). polypeptide, polygenic mRNA; polynucleotides The mRNA becomes attached to cytoplasmic with random sequence (e.g., a polymer containing ribonucleoprotein particles (ribosomes) which are adenylate, uridylate, and guanylate units), poly the sites of protein synthesis, and there it deter- (A, U, G). Trinucleotide codons are shown by mines the order of linkage of amino acids into a base initials (e.g., ApUpG, AUG). specific protein (translation) (17, 243, 291). The mRNA is translated in the 5' to 3' direction (291). Cell-free Protein Synthesizing Systems The synthesis of a protein is initiated at the In vitro systems have been a major tool for amino-terminal amino acid and proceeds towards examining the mechanism of protein biosynthesis. the carboxy-terminal amino acid (17, 32a, 84a, They can be prepared (277) by disintegrating cells 243, 291). During translation, a group of three in aqueous media, removing unbroken cells and adjacent nucleotides in the mRNA (codon) speci- cell debris by low-speed centrifugation and small fies which amino acid is to be linked to the grow- molecules by dialysis. To observe protein syn- ing peptide chain. It has been established which thesis with such extracts, one requires the addition codons specify each of the 20 amino acids (74). of adenosine triphosphate (ATP), guanosine tri- (This is the genetic code). It appears that the phosphate (GTP), an ATP-generating system, sequence of amino acids in a polypeptide chain proper ions (Mg++ and either K+ or NH4+), contains all of the information required for sulfhydryl compounds (which were found to sta- generating the three-dimensional structure of the bilize the system), and amino acids (some of native protein molecule (chain folding) (13). The which are usually radioactively labeled). In such topic of this review is restricted to certain aspects a system, mRNA can be translated into protein. of the mechanism of translation, mainly, al- The translation is assayed by following the in- though not exclusively, as elucidated in microor- corporation of labeled amino acids into protein. ganisms. The regulation of protein synthesis (99) The messenger may be present in the extract is not discussed. A collection of significant in- (endogenous messenger), or it may be added vestigations on mammalian protein synthesis was (exogenous messenger). (In the latter case, the presented in a volume dedicated to the memory endogenous messenger is usually inactivated by of R. Schweet (14). Similar studies in plant sys- incubating the extract before the amino acid in- tems have been described recently (5). corporation experiment to provide time for the nucleases in the extract to degrade the endogenous Abbreviations mRNA.) The exogenous messenger can be either Shorthand writing of oligonucleotides and poly- a natural or a synthetic polyribonucleotide. The nucleotides and abbreviations for nucleotides, use of synthetic polyribonucleotides of known amino-acid residues, etc., are as recommended in composition or sequence was of utmost signifi- J. Biol. Chem. 241:527 (1966). cance in deciphering the genetic code (214, 279, In addition to those identified in the text, ab- 280). breviations are used as follows: RNA capable of The cell extract can be further fractionated by accepting, for example, glycine, tRNAGlY; amino- centrifugation at high speed. The resulting
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