Role Of Guanosine Triphosphate Research Articles

Role of Guanosine Triphosphate in Ferric Ion-Linked Fenton Chemistry

We measured the production of reactive hydroxyl radical (OH.) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3-carboxylic acid (3-CCA) reacts with the OH. produced by Fe2+, Fe3+ or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe(2+)-linked OH. as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe(2+)-linked production of OH.. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce OH. with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of OH.. However, the Fe(2+)-mediated yield of OH. is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for OH. and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of OH. by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe(2+)-generated OH.. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with OH.. The maximal production of reactive OH., compared to all nucleotides and phosphates tested, occurs with a ratio of 2 TPP/Fe2+ complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated OH.. The TPP/Fe(2+)-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules.

Microtubule Dynamics Modulated by Guanosine Triphosphate Hydrolysis Activity of β-Tubulin

Microtubule dynamic instability underlies many cellular functions, including spindle morphogenesis and chromosome movement. The role of guanosine triphosphate (GTP) hydrolysis in dynamic instability was investigated by introduction of four mutations into yeast beta-tubulin at amino acids 103 to 109, a site thought to participate in GTP hydrolysis. Three of the mutations increased both the assembly-dependent rate of GTP hydrolysis and the average length of steady-state microtubules over time, a measure of dynamic instability. The fourth mutation did not substantially affect the rate of GTP hydrolysis or the steady-state microtubule lengths. These results demonstrate that the rate of GTP hydrolysis can modulate microtubule length and hence dynamic instability.

Binding of aminoacyl-tRNA to ribosomes promoted by elongation factor Tu. Studies on the role of GTP hydrolysis.

The role of guanosine triphosphate (GTP) in the elongation factor Tu(EF-Tu)promoted binding of aminoacyl-tRNA to ribosomes has been investigated. In the presence of EF-Tu and GTP, phenylalanyl-tRNA (Phe-tRNA) was bound to the A site of the poly(U).ribosome complex having N-acetylphenylalanyl-tRNA on its P site. EF-Tu could be utilized repeatedly in this binding reaction in the presence of GTP but was used only once and remained bound to ribosomes when GTP was replaced by 5'guanylyl methylenediphosphonate (Gpp(CH2)p), a nonhydrolyzable analog of GTP. The binylalanyl-tRNA, while little dipeptide was formed in the latter case. However, when EF-Tu and Gpp(CH2)p bound to the ribosomal complex were released by centrifugation through 10% sucrose containing 0.2 mM GDP, the yield of the dipeptide was correspondingly increased. It was concluded that the role of GTP in this reaction is to facilitate the transfer of Phe-tRNA to ribosomes by virtue of the high affinity of EF-Tu.GTP for ribosomes. The subsequent conversion of EF-Tu.GTP to EF-Tu.GDP, a form of EF-Tu with low affinity for ribosomes as well as for Phe-tRNA, resulted in the detachment of EF-Tu. Thus, the hydrolysis of GTP seems to be required for the release of EF-Tu from ribosomes, which is necessary for peptidyl transfer and the reutilization of EF-Tu. A freely reversible interaction between ribosome and Phe-tRNA.EF-Tu.Gpp(CH2)p complex was also demonstrated.

The Role of Guanosine Triphosphate in Translocation Reaction Catalyzed by Elongation Factor G

Abstract The role of GTP in the translocation reaction catalyzed by elongation factor G (EF-G) has been investigated. The addition of EF-G and GTP to a poly(U)-ribosome complex having deacylated tRNA in the P site and N-acetyldiphenyl-alanyl-tRNA in the A site (Complex II) results in the shift of N-acetyldiphenylalanyl-tRNA from the A to the P site of ribosomes with a concomitant release of tRNA from the P site. It was found that 5'-guanylylmethylenediphosphonate (Gpp(CH2)p), a nonhydrolyzable analog of GTP, could substitute for GTP in this reaction provided that a stoichiometric amount of EF-G is employed. When Complex II was incubated with a stoichiometric amount of EF-G and Gpp(CH2)p, the deacylated tRNA was released from the P site and the N-acetyldiphenylalanyl-tRNA in the A site was shifted to the P site to become puromycin-reactive. It is concluded that the role of GTP in this reaction is to facilitate the binding of EF-G to Complex II. The translocation reaction is promoted probably by conformational change of Complex II induced through interaction with EF-G and GTP. The hydrolysis of GTP appears to be required for the removal of EF-G after completion of the translocation reaction.

Studies on the Role of Guanosine Triphosphate in Polypeptide Chain Initiation in Escherichia coli

Abstract 1. The formation and properties of the 30 S initiation complex and its subsequent conversion to the 70 S initiation complex have been studied. The 30 S polypeptide chain initiation complex contains fMet-tRNA and intact GTP in equimolar amounts. Incorporation of both species into the complex requires mRNA (f2 RNA, poly (U, G), or A-U-G) and large amounts of initiation factor IF 2. Initiation factor IF 1 stimulates 30 S complex formation. 2. Addition of 50 S subunits to the isolated 30 S complex results in the hydrolysis of all bound GTP to GDP and Pi. In the absence of 50 S subunits, no hydrolysis occurs. Concurrently a 70 S couple is formed and fMet-tRNA in the complex becomes available for peptidyl transfer. 3. Under appropriate conditions the 30 S complex can be depleted of its complement of GTP while retaining a full complement of fMet-tRNA. GTP released in this manner remains intact. Such a GTP-deficient 30 S complex can still accept 50 S subunits to form a 70 S couple and can still donate fMet into peptide linkage. 4. At the low (catalytic) levels of factor IF 2 necessary for GTP-dependent 70 S initiation complex formation using a mixture of ribosomal subunits, 5'-guanylyl methylene diphosphonate (GMP-PCP) does not replace GTP. At the high (stoichiometric) levels of IF 2 necessary for GTP-dependent 30 S initiation complex formation, GMP-PCP can substitute for GTP in promoting 30 S complex formation. The GMP-PCP-stimulated 30 S complex can then accept a 50 S subunit to form a 70 S couple. However, this complex is inactive in subsequent peptidyl transfer. 5. A model for the initiation process is presented which suggests that a GTP-dependent translocation of fMet-tRNA does not occur. Rather, it is postulated that fMet-tRNA binds directly to the donor site equivalent on the 30 S subunit. Upon 50 S addition, a 70 S couple is formed. GTP hydrolysis serves to release IF 2 and GTP from the initiation complex. Released IF 2 recycles to catalyze another round of initiation. Release of GTP and IF 2 also serves to unblock fMet-tRNA for peptidyl transfer.

Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. The role of guanosine triphosphate.

1. Transferase I from rat liver binds relatively low quantities of GTP when incubated with this nucleotide in the absence of aminoacyl-tRNA. 2. Transferase I reacts with both aminoacyl-tRNA and GTP to form a relatively stable complex that is retained on cellulose nitrate filters. The ternary complex transferase I-aminoacyl-tRNA-GTP is also formed when the transferase I-aminoacyl-tRNA complex is incubated with GTP or during the incubation of the transferase I-GTP complex with aminoacyl-tRNA. Synthesis of this complex does not require the presence of Mg(2+). 3. In the presence of Mg(2+) the ternary complex becomes readily bound to ribosomes without requirements for any other cofactors. 4. An extensive cleavage of GTP takes place when aminoacyl-tRNA becomes bound to ribosomes. 5. The low interdependence of reactions leading to the formation of transferase I complexes with aminoacyl-tRNA and GTP indicates that the mechanisms of the binding reaction in mammalian systems may be different from those in bacterial cells.