Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5-terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m 7 GpppG, the rate constant of association, k on , was dependent on ionic strength, decreasing progressively up to 350 mM KCl, but the rate constant of dissociation, k off , was independent of ionic strength. Phosphorylation of eIF4E decreased k on by 2.1-2.3-fold at 50 -100 mM KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mM. Contrary to published evidence, eIF4E phosphorylation had no effect on k off . Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single-to double-exponential as the eIF4E concentration was increased. However, measuring k off for dissociation of a pre-formed eIF4E⅐m 7 GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in k off . This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.The efficiency of mRNA translational initiation is strongly enhanced by the 5Ј-terminal cap, m 7 GpppN (1). The cap specifically binds to eIF4E, 2 which may be the first canonical initiation factors to interact with mRNA during its recruitment to the ribosome. eIF4E in turn binds to eIF4G, a protein that also interacts with the RNA helicase eIF4A to promote unwinding of mRNA secondary structure, with the multisubunit factor eIF3 to recruit the 43 S initiation complex, and with the cytoplasmic poly(A)-binding protein to enhance initiation of poly(A)-containing mRNAs. Initiation codon recognition is followed by dissociation of eIFs and joining of the 60 S ribosomal subunit to form the elongation-competent 80 S initiation complex. eIF4E has been extensively investigated in organisms that range from yeast to mammals (2-7). Besides translation, eIF4E also functions in nucleocytoplasmic transport of mRNA, sequestration of mRNA in a nontranslatable state, and stabilization of mRNA against decay in the cytosol (8 -10). The three-dimensional structures of human, mouse, and Saccharomyces cerevisiae eIF4E have been solved (11-13). The complex of full-length human eIF4E with m Cap analogs bind to eIF4E in a tight complex, a step that has been studied primarily by equilibrium techniques (15-33). Intrinsic Trp fluorescence quenching of N-termi...

SummaryWe discuss recent experiments that have illuminated individual steps in the reaction cycle of the Escherichia coli Hsp70 molecular chaperone DnaK. Using this new information, we compare two distinctly different global mechanisms of action -holding versus unfolding -and argue that the available evidence suggests that DnaK is an unfoldase.

The mechanism of the ATPase cycle of the 70-kDa Escherichia coli molecular chaperone DnaK was investigated by following ATP-induced changes in the tryptophan fluorescence of DnaK. Three steps in the cycle were investigated. (i) Stopped-flow experiments revealed that ATP induces a biphasic reduction in the tryptophan fluorescence of DnaK. The rate of the fast fluorescence transition exhibited a hyperbolic dependence on the ATP concentration, with a maximum rate equal to 56 (+/- 10) s-1 at 35 degrees C, whereas the rate of the slow fluorescence transition was nearly independent of the ATP concentration (4.2 +/- 0.2 s-1). These results are consistent with the three-step sequential reaction E + ATP<-->E-ATP<-->E*-ATP<-->E**-ATP prior to DnaK-catalyzed ATP hydrolysis, where the formation of a collisional complex (E-ATP) causes no change in fluorescence but is followed by two first-order transitions that reduce the fluorescence. (ii) The kinetics of ADP replacement from preformed DnaK-ADP complexes by ATP followed simple exponential kinetics, kADP = 0.038 (+/- 0.002) s-1 at 35 degrees C. The ADP off rate was reduced approximately 10-fold by inorganic phosphate (20 mM). (iii) Single-turnover experiments ([DnaK] = [ATP] = 1 microM) revealed a slow, first-order increase in tryptophan fluorescence [k(obs) = 0.0015 (+/- 0.0001) s-1, 37 degrees C] that was identical to the rate of DnaK-catalyzed ATP hydrolysis [k(hy) = 0.0014 (+/- 0.0001) s-1, 37 degrees C]. This slow increase in fluorescence is consistent with a E**-->E conformational transition. A model for the ATPase cycle of DnaK is proposed in which ATP has two distinct functions: ATP binding to the ATPase domain triggers two conformational transitions in a chaperone molecule, and ATP hydrolysis--the slow step in the reaction cycle--reverses the transitions.

Helicobacter pylori persistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a few H. pylori adhesins have been paired with targets in host tissue. The alpAB locus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation of H. pylori AlpA and AlpB reduces binding of H. pylori to laminin while expression of plasmid-borne alpA or alpB confers laminin-binding ability to Escherichia coli. An H. pylori strain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute to H. pylori laminin binding. Contrary to expectations, the H. pylori SS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring during H. pylori infection.