The rate of cleavage of GTP on the binding of Phe-tRNA.elongation factor Tu.GTP to poly(U)-programmed ribosomes of Escherichia coli.

The tubulin concentration dependence of the rates of microtubule elongation and accompanying GTP hydrolysis has been studied over a large range of tubulin concentration. GTP hydrolysis followed the elongation process closely at low tubulin concentration and became gradually uncoupled at higher concentrations, reaching a limiting rate of 35-40 s-1. The kinetic parameters for microtubule growth were different at low and high tubulin concentrations. Elongation of microtubules has also been studied in solutions containing GDP and GTP in variable proportions. Only traces of GTP present in GDP were necessary to confer a high stability (low critical concentration) to microtubules. Pure GDP-tubulin was found unable to elongate microtubules in the absence of GTP but blocked microtubule ends with an equilibrium dissociation constant of 5-6 microM. These data were accounted for by a model within which, in the presence of GTP-tubulin at high concentration, microtubules grow at a fast rate with a large GTP cap; the GTP cap may be quite short in the region of the critical concentration; microtubule stability is linked to the strong interaction between GTP and GDP subunits at the elongating site; dimeric GDP-tubulin does not have the appropriate conformation to undergo reversible polymerization. These results are discussed with regard to possible role of GDP and GTP and of GTP hydrolysis in microtubule dynamics.

Section: Discussionsupporting

confidence: 63%

Microtubule elongation and guanosine 5'-triphosphate hydrolysis. Role of guanine nucleotides in microtubule dynamics

Carlier

Didry²,

Pantaloni³

1987

“…For each time point, 15 µ (30 pmol) of poly(U)programmed 70S ribosome containing Ar-acetyl[l4C]Phe-tRNAPhe, prepared as described (Thompson et al, 1981), was mixed with 5 µ (2.5 pmol) of the ternary complex containing modified or unmodified [3H]Phe-tRNAPhe (45-60 Ci/mmol) and [ -32 ] (40-60 Ci/mmol) in buffer B at 5 °C. The reaction was terminated by the addition of 10 µ of 0.5 M EDTA, and 12-µ aliquots were analyzed for GTP hydrolysis and TV-acetyl [14C] phenylalanyl [3H] phenylalanine by previously described procedures (Eccleston et al, 1985;Thompson et al, 1986). Rate constants for GTP hydrolysis (*gtp) and dipeptide formation (fcPEP) were determined by computer simulation of the reaction progress curves (Thompson et al, 1980), taking into account the concentrations of active ribosomes and the ternary complex.…”

Section: Methodsmentioning

confidence: 99%

In vitro analysis of translational rate and accuracy with an unmodified tRNA

Harrington¹,

Nazarenko²,

Dix³

et al. 1993

Escherichia coli tRNA(Phe) transcript lacking all the modified nucleosides was investigated in an in vitro translation system. To estimate the affinity of tRNA toward EF-Tu, Kd and K-1 were measured by the nuclease protection assay, and it was shown that the absence of modifications decreases ternary complex stability less than 2-fold. The activity of unmodified Phe-tRNA(Phe) on E. coli ribosomes was compared to modified Phe-tRNA(Phe) using the framework of the kinetic proofreading mechanism (Thompson & Dix, 1982) with both cognate and noncognate codons. Values of the individual rate constants in the elongation process showed that the modifications increased the accuracy of translation by (1) decreasing the rate of dipeptide synthesis and (2) increasing the rate of rejection with noncognate codons.

“…Quenched-flow experiments were performed on the instrument described by Eccleston et al (1985) except that reactants were loaded into both syringes rather than the final sample loop. A 200-µ total reaction mixture was ejected into 200 µ of 10% perchloric acid at 4 °C and rapidly adjusted to pH 4 by the addition of 100 µ of 4 M sodium acetate.…”

Section: Methodsmentioning

confidence: 99%

Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: Characterization of two acto.cntdot.S1.cntdot.ADP complexes

Woodward¹,

Eccleston²,

Geeves³

1991

148

129

We have used a fluorescent analogue of ATP, mantATP [2'(3')-O-(N-methylanthraniloyl)-adenosine 5'-triphosphate; Hiratsuka T. (1983) Biochim. Biophys. Acta 742, 496-508], and made a detailed kinetic study of the interaction of mantATP and mantADP with S1 and acto-S1. We have shown that these analogues behave like ATP and ADP, respectively. In addition, we have demonstrated that this analogue can distinguish between two acto-S1 complexes, the A-M.N (attached) and A.M.N (rigor-like) states [Geeves, M. A., Good, R. S., & Gutfreund, H. (1984) J. Muscle Res. Cell Motil. 5, 351-361]. Previously, these two states were observed with a pyrene label on Cys 374 of actin. This isomerization can now be monitored at two spatially distinct sites on the ternary complex, indicative of a major conformational change in the ternary complex. Also, we have measured the rate of ADP dissociation from both A-M.N and A.M.N directly and shown these to differ by a factor of 1000. Thus the results presented here support the model of Geeves et al. and are consistent with the A-M.N to A.M.N transition being coupled to the force-generating event of the crossbridge cycle.