GTP in protein synthesis

Cells use GTP and CTP, as well as UTP and ATP, to form activated intermediates. Different anabolic pathways generally use different nucleotides as their direct source of high phosphate bond energy UTP is used for combining sugars, CTP in lipid synthesis, and GTP in protein synthesis. 

Ertel, R., Redfield, B. and Weissbach, H. (1968) Role of GTP in protein synthesis interaction of GTP with soluble transfer factors from E. coli. Arch. Biochem. Biophys. 128, 331-338. 

GTP is an essential component in protein synthesis, and it has been shown recently that another guanosine polyphosphate, guanosine 3, 5 -... 

Energy requirements in protein synthesis are high. Four energy-rich phosphoric acid anhydride bonds are hydrolyzed for each amino acid residue. Amino acid activation uses up two energy-rich bonds per amino acid (ATP AMP + PP see p. 248), and two GTPs are consumed per elongation cycle. In addition, initiation and termination each require one GTP per chain. 

Energy consumption in protein synthesis The energy consumed by the process is the guarantee of fidelity of protein synthesis. Four molecules (or more) of GTP (ATP equivalent) are required for the synthesis of each peptide bond. 

Overview of reactions in protein synthesis. (aab aa2, aa3 = amino acids l, 2, 3.) Protein synthesis requires transfer RNAs for each amino acid, ribosomes, messenger RNA, and a number of dissociable protein factors in addition to ATP, GTP, and divalent cations. First the transfer RNAs become charged with amino acids, then the initiation complex is formed. Peptide synthesis does not start until the second aminoacyl tRNA becomes bound to the ribosome. Elongation reactions involve peptide bond formation, dissociation of the discharged tRNA, and translocation. The elongation process is repeated many times until the termination codon is reached. Termination is marked by the dissociation of the messenger RNA... 

In at least one eukaryote, Tetmhymem, the pre-rRNA molecule contains an intron. Removal of the intron during processing of the pre-rRNA does not require the assistance of any protein Instead, in the presence of guanosine, GMP, GDP or GTP, the intron excises itself, a phenomenon known as selfsplicing. This was the first demonstration of ribozymes, that is, catalytic RNA molecules that catalyze specific reactions. The list of ribozymes is growing. For example, self-splicing introns have been discovered in some eukaryotic mRNAs and even peptidyl transferase, a key enzyme activity in protein synthesis, is now known to be a ribozyme (see Topic H2). 

The aminoacyl transfer reaction, one of the latter stages in protein synthesis, involves incorporation of amino acids from soluble ribonucleic acid-amino acid into ribosomal protein. This reaction requires guanosine triphosphate and a soluble portion of the cell. Evidence has been obtained with rat liver preparations that aminoacyl transfer is catalyzed by two protein factors, aminoacyl transferases (or polymerases) I and n, which have been resolved and partially purified from the soluble fraction. Transferase n activity has also been obtained from deoxycholate-soluble extracts of microsomes. With purified transferases I and n, incorporation is observed with relatively low levels of GTP its sulfhy-dryl requirement is met by a variety of compounds. The characteristics of this purified amino acid incorporating system, in terms of dependency on the concentration of its components, are described. 

Guanosine triphosphate (GTP) is required by which of the following steps in protein synthesis ... 

The possible role of guanosine 3 -pyrophosphate-5 -triphosphate (30) in protein synthesis in E. coli has been examined. Although it can substitute for GTP reactions catalysed by initiation factor (IF)2 and elongation factor... 

Experiments show that it is on the ribosomes that protein synthesis actually occurs. If cells synthesizing protein from radioactive amino acids are studied, the radioactivity is first found bound to the ribosomes, and is only later released from them as soluble protein. A cell suspension from which ribosomes have been removed can never be made to synthesize protein, whilst if they are subsequently replaced synthesis can proceed rapidly. But the ribosomes alone are inadequate. In order to incorporate radioactive amino acids into new protein there needs to be added to the ribosomes a preparation of soluble cell material which contains certain enzymes, the soluble low-molecular-weight messenger RNA (m-RNA), transfer RNA (t-RNA), ATP, GTP, and ions like magnesium and potassium. So what role do these various substances perform in protein synthesis ... 

Enzymes involved in protein synthesis also possess potential, e.g. aminoacyl-tRNA-synthetase-aminoadenylate complex 1791, the arginyl-tRNA protein arginyl-transferase11801, and nonribosomal poly- or multienzyme complexes1721 which require ATP or GTP to activate the carboxyl group of an amino acid, and seem to accept various amino acid nucleophiles for peptide bond formation. Flowever, the application of these enzyme systems for generally practical peptide bond formation is rather limited. 

The ot subunits of G proteins are part of a family of small GTP-binding proteins that are active when GTP is bound and inactive in the presence of GDP. This includes the Ras oncogene proteins (see here) and the GTP-binding elongation factors in protein synthesis (see here). 

Our calculated values for the actual rate of incorporation are based on the assumption that the total acid-soluble GTP pool is used as a source of nucleotides for RNA synthesis i.e., precursor pools are not compartmentalized. However, in a series of studies on tissue culture cells, Plagemann (1971a,b 1972) has reported that most exogenous nucleosides enter and expand a precursor pool not used directly for RNA synthesis. Likewise, Ecker (1972) has reported that the amino acid pools active in protein synthesis in Ram pipiens oocytes are con-... 

GTP is also essential for polypeptide synthesis in cell-free systems. The mechanism for the GTP utilization in protein synthesis is not yet clear, although GTP may supply the energy needed for the coordinated reactions involving the binding of amino acyl-tRNA, formation of the peptide bond and the movement of messenger RNA along ribosomes. 

The fate of EF-G after translocation is not clear, but the protein is believed to be released and recycled. The role of GTP hydrolysis in protein synthesis remains unknown. To date, no functional phosphorylation or guanylation of any of the factors involved has been described. 

As already stated DNA itself is not directly involved in protein synthesis. Instead it is the mRNAs which define the amino acid sequence during protein synthesis. This assembly of amino acids on an mRNA template, to form a polypeptide, is an extremely complex process and is known as translation. It requires many factors including the amino acids, mRNA, ribosomes, a whole series of different tRNAs, many enzymes, protein factors, ATP, GTP and Mg. ... 

In protein synthesis, it is the conformation of the functional proteins associated with GTP (E-GTP) which is utilized to drive the reaction. The factor is then released from ribosomes as E-GDP, and the exogenous energy is fed into this system to regenerate E-GDP to E-GTP. In other energy-transducing systems, similar but variant types of conformational transition are naturally observed. For example, in active transport, two kinds of conformational transitions could be found, namely E-ATP and E P, and E P and E. It might be assumed that the former transition is utilized for the transport of a cation from outside to inside of the membrane and the latter transition is utilized to the counter-transport of another cation to the opposite direction. 

Subsequently, other investigators established that the release of some initiation and termination factors from the ribosome also depends on GTP cleavage. Further insight into the role of GTP and its cleavage in protein synthesis has been provided by the studies of... 

The interaction of the ternary complex with the ribosome [eqn. (7)] is extremely complicated, for it involves not only recognition of the AA-tRNA in the complex by the proper codon on the mRNA, but also hydrolysis of GTP. GTP hydrolysis occurs at three different stages in protein synthesis and will be described in further detail below. The hydrolysis of GTP in reaction (7) has been demonstrated by several groups,and was of immediate interest to workers in the field, for it seemed ideally suited to study the mechanism of at least one GTP hydrolytic reaction. The substrate, that is the ternary complex, is readily prepared (even with three labels and relatively stable. The reaction could... 

Fusidic acid inhibits EFG activity [eqn. (10)] since the stable fusidic acid complex prevents EFG from acting catalytically in translocation. A tight binding of EFG to the ribosome can also be achieved with GDPCP. In this case a SOS subunit-EFG-GDPCP complex is formed. The data indicate that since hydrolysis does not occur with this analog, EFG cannot recycle and translocation is inhibited. In retrospect, the studies with fusidic acid have focused our thinking on the overall role of GTP hydrolysis in protein synthesis. 

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