ppGpp inhibition of elongation factors Tu, G and Ts during polypeptide synthesis

Summary The inhibition of elongation factors G, Tu and Ts by ppGpp was studied in vitro in a translation system with missense frequency and elongation rate similar to those in vivo. ppGpp inhibits EF-G with K_I=6x10^-5 M. When ppGpp is in twofold excess over GTP and EF-G is the rate-limiting component, the elongation rate is reduced two-fold by ppGpp. EF-Tu is inhibited with K_I=7x10^-7 M in the absence of EF-Ts. When EF-Ts is added, the binding of ppGpp to EF-Tu becomes successively weaker. 1/K_I depends linearly on 1/[Ts] and the intercept at the abscissa gives K_I=4x10^-5 M. This reflects the binding of ppGpp to the binary TuTs complex. The slope reveals that the binding of EF-Ts to the TuMS binary complex is strong (10^-6 M). ppGpp may thus inhibit the cycling of EF-Tu indirectly by the removal of the free EF-Ts by its adsorption to TuMS, as well as directly by simple binding to Tu. EF-Tu inhibition by ppGpp can be fully reversed by high levels of aminoacyl-tRNA only in the presence of EF-Ts and at low ribosomal activity. Our in vitro observations have been extrapolated to in vivo conditions with conclusions as follows: Under strong amino acid starvation ppGpp in two-fold excess over GTP cannot reduce significantly the elongation rate of ribosomes and thereby restore the errors to their normal levels as in the stringent response. Under weak starvation, in contrast, a significant rate reduction can be achieved by the trapping of EF-Ts in complex with TuppGpp.     

Effect of Thermus thermophilus elongation factor Ts on the conformation of elongation factor Tu

Affinity labeling in situ of the Thermus thermophilus elongation factor Tu (EF-Tu) nucleotide binding site was achieved with periodate-oxidized GDP (GDPoxi) or GTP (GTPoxi) in the absence and presence of elongation factor Ts (EF-Ts). Lys52 and Lys137, both reacting with GDPoxi and GTPoxi, are located in the nucleotide binding region. In the absence of EF-Ts Lys137 and to a lesser extent Lys52 were accessible to the reaction with GTPoxi. GDPoxi reacted much more efficiently with Lys52 than with Lys137 under these conditions [Peter, M. E., Wittman-Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132-9138]. In the presence of EF-Ts, GDPoxi reacted more efficiently with Lys137 than with Lys52, indicating that the interaction of EF-Ts with EF-Tu.GDPoxi induces a conformation resembling that of the EF-Tu.GDPoxi complex in the absence of EF-Ts. Binding of EF-Ts to EF-Tu.GDP enhances the accessibility of the Arg59-Gly60 peptide bond of EF-Tu to trypsin cleavage. Hydrolysis of this peptide bond does not interfere with the ability of EF-Ts to bind to EF-Tu. EF-Ts is protected against trypsin cleavage by interaction with EF-Tu.GDP. High concentrations of EF-Ts did not interfere significantly with aminoacyl-tRNA.EF-Tu.GTP complex formation.     

Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and elongation factor Ts

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu.GTP.aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu.GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu.Ts). Mammalian mitochondrial EF-Tu (EF-Tu(mt)) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tu(mt) subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K(tRNA)) and for the dissociation of the EF-Tu.Ts(mt) complex (K(Ts)) were investigated. The equilibrium dissociation constant for the ternary complex was 18 +/- 4 nm, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 x 10(-)(4) s(-)(1), which is essentially equivalent to that observed for the ternary complex in Escherichia coli. The binding of EF-Tu(mt) to EF-Ts(mt) is mutually exclusive with the formation of the ternary complex. K(Ts) was determined by quantifying the effects of increasing concentrations of EF-Ts(mt) on the amount of ternary complex formed with EF-Tu(mt). The value obtained for K(Ts) (5.5 +/- 1.3 nm) is comparable to the value of K(tRNA).     

Bovine mitochondrial protein synthesis elongation factors. Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex

Animal mitochondrial protein synthesis factors elongation factor (EF) Tu and EF-Ts have been purified as an EF-Tu.Ts complex from crude extracts of bovine liver mitochondria. The mitochondrial complex has been purified 10,000-fold to near homogeneity by a combination of chromatographic procedures including high performance liquid chromatography. The mitochondrial EF-Tu.Ts complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides. No guanine nucleotide binding to this complex can be observed in the standard nitrocellulose filter binding assay. Mitochondrial EF-Ts activity can be detected by its ability to facilitate guanine nucleotide exchange with Escherichia coli EF-Tu. The EF-Tumt exhibits similar levels of activity on isolated mammalian mitochondrial and E. coli ribosomes, but displays minimal activity on Euglena gracilis chloroplast 70 S ribosomes and has no detectable activity on wheat germ cytoplasmic ribosomes. In contrast to the bacterial EF-Tu and the EF-Tu from the chloroplast of E. gracilis, the ability of the mitochondrial factor to catalyze polymerization is not inhibited by the antibiotic kirromycin.     

Novel data on interactions of elongation factor Ts.

Interactions of EF-Ts with EF-Tu at all steps of the elongation cycle were studied by limited trypsinolysis, gel-filtration, analytical centrifugation and fluorescence polarization techniques. It is shown that EF-Ts does not dissociate from EF-Tu after GDP to GTP exchange, but remains bound to the Aa-tRNA.EF-Tu.GTP complex up to GTP hydrolysis stage on the ribosome. The possible role of these interactions is discussed.     

Bacterial elongation factor Ts: isolation and reactivity with elongation factor Tu.

An improved method for the purification of bacterial polypeptide elongation factor Ts (EF-Ts) from one mesophile (Escherichia coli) and two thermophiles (Bacillus stearothermophilus and PS3) is described. The improvements are both in the facility of isolation and in increased yields. The purified factors were used for cross-reactivity studies with elongation factor Tu (EF-Tu) obtained from the same bacterial strains. In all combinations studied, the efficiency of EF-Ts in catalyzing the exchange of EF-Tu-bound GDP was proportional to the strength of the protein-protein complex. Whereas the factors from the two thermophiles were interchangeable, the mesophilic EF-Ts formed a very weak complex with thermophilic EF-Tu; however, thermophilic EF-Ts formed very strong complexes with mesophilic EF-Tu. Thus, e.g., EF-Tu from E. coli formed a complex with EF-Ts from B. stearothermophilus which was 10 times more stable than the corresponding homologous complex.     

Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts

The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP and elongation factor Ts (EF-Ts) have been investigated. The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation at 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of EF-Tu. The fluorescence lifetime of this tryptophan residue in both complexes was investigated by using a multifrequency phase fluorometer which achieves a broad range of modulation frequencies utilizing the harmonic content of a mode-locked laser. These results indicated a heterogeneous emission with major components near 4.8 ns for both complexes. Quenching experiments on both complexes indicated limited accessibility of the tryptophan residue to acrylamide and virtually no accessibility to iodide ion. The quenching patterns exhibited by EF-Tu-GDP and EF-Tu X EF-Ts were, however, different; both quenchers were more efficient at quenching the emission from the EF-Tu x EF-Ts complex. Steady-state and dynamic polarization measurements revealed limited local mobility for the tryptophan in the EF-Tu x GDP complex whereas formation of the EF-Tu x EF-Ts complex led to a dramatic increase in this local mobility.     

Functional effects of deleting the coiled-coil motif in Escherichia coli elongation factor Ts.

Elongation factor Ts (EF-Ts) is the guanine nucleotide-exchange factor for elongation factor Tu (EF-Tu) that is responsible for promoting the binding of aminoacyl-tRNA to the mRNA-programmed ribosome. The structure of the Escherichia coli EF-Tu-EF-Ts complex reveals a protruding antiparallel coiled-coil motif in EF-Ts, which is responsible for the dimerization of EF-Ts in the crystal. In this study, the sequence encoding the coiled-coil motif in EF-Ts was deleted from the genome in Escherichia coli by gene replacement. The growth rate of the resulting mutant strain was 70-95% of that of the wild-type strain, depending on the growth conditions used. The mutant strain sensed amino acid starvation and synthesized the nucleotides guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate at a lower cell density than the wild-type strain. Deletion of the coiled-coil motif only partially reduced the ability of EF-Ts to stimulate the guanine nucleotide exchange in EF-Tu. However, the concentration of guanine nucleotides (GDP and GTP) required to dissociate the mutant EF-Tu-EF-Ts complex was at least two orders of magnitude lower than that for the wild-type complex. The results show that the coiled-coil motif plays a significant role in the ability of EF-Ts to compete with guanine nucleotides for the binding to EF-Tu. The present results also indicate that the deletion alters the competition between EF-Ts and kirromycin for the binding to EF-Tu.     

A study of the kinetic mechanism of elongation factor Ts

Elongation factor Ts (EF-Ts) catalyzes the reaction EF-Tu X GDP + nucleotide diphosphate (NDP) reversible EF-Tu X NDP + GDP where NDP is GDP, IDP, GTP, or GMP X PCP. The EF-Ts-catalyzed exchange rates were measured at a series of concentrations of EF-Tu X [3H] GDP and free nucleotide. Plotting the rate data according to the Hanes method produced a series of lines intersecting on the ordinate, a characteristic of substituted enzyme mechanisms. GDP is a competitive inhibitor of IDP exchange, a result predicted for the substituted enzyme mechanism but inconsistent with ternary complex mechanisms that involve an intermediate complex containing EF-Ts and both substrates. The exchange of both GTP and the GTP analog GMP X PCP also follow the substituted enzyme mechanism. The maximal rates of exchange of GDP and GTP are the same, which indicates that the rates of dissociation of EF-Ts from EF-Tu X GDP and EF-Tu X GTP are the same. The steady-state maximal exchange rate is slower by a factor of 20 than the previously reported rate of dissociation of GDP from EF-Ts X EF-Tu. This is interpreted to mean that the rate-determining step in the exchange reaction is the dissociation of EF-Ts from EF-Tu X GDP.     

A mutant of Escherichia coli blocked in peptide elongation: altered elongation factor Ts

Among our transfer RNA-dependent growth mutants, one, HAK88, was found that carries an altered elongation factor Ts. The activity of mutant EFTs to bind GDP to EFTu, or to form the ternary complex (aminoacyl-tRNA-EFTu-GTP) is thermolabile. The effect of magnesium on the formation of EFTu-GDP from the EFTu-EFTs complex of HAK8 shows that a four to fivefold increase of the duplex formation occurs when the magnesium concentration is increased from 10^?6m to 10^?2m at 0 °C and at 41 °C. However, at higher temperatures, formation of the binary EFTu-GDP from the EFTu-EFTs complex of HAK88 is depressed, even at 10^?3m to 10^?2m-magnesium. The binding of GDP to the wild-type or mutant EFTu-EFTs complex at 0 °C and 42 °C indicates that the formation of EFTu-GDP is inhibited at 42 °C only when mutant complex is used for the assay. Binding of GTP to complete bacteriophage Qβ replicase (which is known to contain EFTs) formed in phage-infected HAK88 is also inhibited at 42 °C.     

Eucaryotic elongation factors Ts is an integral component of rabbit reticulocyte elongation factor 1.

Elongation factor 1 (EF-1) was purified from rabbit reticulocytes and found to contain at least two distinct polypeptides: one of Mr 53 000 and one of Mr 30 000. The 30 000-Mr polypeptide was purified from EF-1 by treatment of the factor with 5.4 M guanidine . HCl and subsequent chromatography on DEAE-BioGel A in the presence of 5 M urea. By a number of functional criteria, the 30 000-Mr polypeptide was found to be the eucaryotic elongation factor Ts (eEF-Ts). These criteria include the ability of the polypeptide to stimulate Artemia salina eEF-Tu-dependent binding of aminoacyl-tRNA to 80-S ribosomes as well as eEF-Tu + EF-2-dependent polyphenylalanine synthesis. The reticulocyte factor also markedly increased the rate of exchange of eEF-Tu . gdp complexes with free GTP. Furthermore, rabbit antibodies to EF-1 from A. salina which was previously shown to contain eEF-Ts [Slobin, L. I. and M?ller, W. (1978) Eur. J. Biochem. 84, 69--77] were found to cross-react with reticulocyte eEF-Ts, suggesting extensive structural homology between brine shrimp and rabbit eEF-Ts. The demonstration that eEF-Ts is and integral component of EF-1 from such diverse sources as brine shrimp and rabbit reticulocytes supports the conclusion that the factor is universally present in eucaryotic EF-1.     

The identification of a domain in Escherichia coli elongation factor Tu that interacts with elongation factor Ts.

A method has been developed to search for the elongation factor Tu (EF-Tu) domain(s) that interact with elongation factor Ts (EF-Ts). This method is based on the suppression of Escherichia coli EF-Tu-dominant negative mutation K136E, a mutation that exerts its effect by sequestering EF-Ts. We have identified nine single-amino acid- substituted suppression mutations in the region 146-199 of EF-Tu. These mutations are R154C, P168L, A174V, K176E, D181G, E190K, D196G, S197F, and I199V. All suppression mutations but one (R154C) significantly affect EF-Tu's ability to interact with EF-Ts under equilibrium conditions. Moreover, with the exception of mutation A174V, the GDP affinity of EF-Tu appears to be relatively unaffected by these mutations. These results suggest that the domain of residues 154 to 199 on EF-Tu is involved in interacting with EF-Ts. These suppression mutations are also capable of suppressing dominant negative mutants N135D and N135I to various degrees. This suggests that dominant negative mutants N135D and N135I are likely to have the same molecular basis as the K136E mutation. The method we have developed in this study is versatile and can be readily adapted to map other regions of EF-Tu. A model of EF-Ts-catalyzed guanine-nucleotide exchange is discussed.     

Qbeta-phage resistance by deletion of the coiled-coil motif in elongation factor Ts

Elongation factor Ts (EF-Ts) is the guanine-nucleotide exchange factor of elongation factor Tu (EF-Tu), which promotes the binding of aminoacyl-tRNA to the mRNA-programmed ribosome in prokaryotes. The EF-Tu.EF-Ts complex, one of the EF-Tu complexes during protein synthesis, is also a component of RNA-dependent RNA polymerases like the polymerase from coliphage Qbeta. The present study shows that the Escherichia coli mutant GRd.tsf lacking the coiled-coil motif of EF-Ts is completely resistant to phage Qbeta and that Qbeta-polymerase complex formation is not observed. GRd.tsf is the first E. coli mutant ever described that is unable to form a Qbeta-polymerase complex while still maintaining an almost normal growth behavior. The phage resistance correlates with an observed instability of the mutant EF-Tu.EF-Ts complex in the presence of guanine nucleotides. Thus, the mutant EF-Tu.EF-Ts is the first EF-Tu.EF-Ts complex ever described that is completely inactive in the Qbeta-polymerase complex despite its almost full activity in protein synthesis. We propose that the role of EF-Ts in the Qbeta-polymerase complex is to control and trap EF-Tu in a stable conformation with affinity for RNA templates while unable to bind aminoacyl-tRNA.     

Crystal structure of the bovine mitochondrial elongation factor Tu.Ts complex

The three-dimensional structure of the bovine mitochondrial elongation factor (EF)-Tu.Ts complex (EF-Tumt.Tsmt) has been determined to 2.2-A resolution using the multi-wavelength anomalous dispersion experimental method. This complex provides the first insight into the structure of EF-Tsmt. EF-Tsmt is similar to Escherichia coli and Thermus thermophilus EF-Ts in the amino-terminal domain. However, the structure of EF-Tsmt deviates considerably in the core domain with a five-stranded beta-sheet forming a portion of subdomain N of the core. In E. coli EF-Ts, this region is composed of a three-stranded sheet. The coiled-coil domain of the E. coli EF-Ts is largely eroded in EF-Tsmt, in which it consists of a large loop packed against subdomain C of the core. The conformation of bovine EF-Tumt in complex with EF-Tsmt is distinct from its conformation in the EF-Tumt.GDP complex. When domain III of bovine EF-Tumt.GDP is superimposed on domain III of EF-Tumt in the EF-Tumt.Tsmt complex, helix B from domain I is also almost superimposed. However, the rest of domain I is rotated relative to this helix toward domain II, which itself is rotated toward domain I relative to domain III. Extensive contacts are observed between the amino-terminal domain of EF-Tsmt and domain I of EF-Tumt. Furthermore, the conserved TDFV sequence of EF-Tsmt also contacts domain I with the side chain of Asp139 contacting helix B of EF-Tumt and inserting the side chain of Phe140 between helices B and C. The structure of the EF-Tumt.Tsmt complex provides new insights into the nucleotide exchange mechanism and provides a framework for explaining much of the mutational data obtained for this complex.     

Kinetics and thermodynamics of the interaction of elongation factor Tu with elongation factor Ts, guanine nucleotides, and aminoacyl-tRNA

The exchange of elongation factor Tu (EF-Tu)-bound GTP in the presence and absence of elongation factor Ts (EF-Ts) was monitored by equilibrium exchange kinetic procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu X GTP X EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu X GTP complex and of GTP to EF-Tu X EF-Ts were calculated to be 7 X 10(7) and 2 X 10(6) M-1, respectively. The dissociation rate constant of GTP from the ternary complex was found to be 13 s-1. This is 500 times larger than the GTP dissociation rate constant from the EF-Tu X GTP complex (2.5 X 10(-2) s-1). A procedure based on the observation that EF-Tu X GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu X GTP with Val-tRNAVal and Phe-tRNAPhe. Binding constants of Phe-tRNAPhe and Val-tRNAVal to EF-Tu X GTP of 4.8 X 10(7) and 1.2 X 10(7)M-1, respectively, were obtained. The exchange of bound GDP with GTP in solution in the presence of EF-Ts was also examined. The kinetics of the reaction were found to be consistent with a rapid equilibrium mechanism. It was observed that the exchange of bound GDP with free GTP in the presence of a large excess of the latter was accelerated by the addition of aminoacyl-tRNA. On the basis of these observations, a complete mechanism to explain the interactions among EF-Tu, EF-Ts, guanine nucleotides, and aminoacyl-tRNA has been developed.     

An improved bulk purification method for Escherichia coli elongation factor, Ts

A bulk purification procedure has been designed to maximize the yield of Escherichia coli elongation factor, Ts, with a minimum of effort and time. The enzyme purification is achieved by DEAE-Sepharose and elongation factor Tu-affinity chromatographies. The typical yield is 150 mg/kg of E. coli (B) cells.     

The kinetic mechanism of the release of nucleotide from elongation factor Tu promoted by elongation factor Ts determined by pressure relaxation studies

The release of a chromophoric analogue of GDP, 2-amino-6-mercaptopurine riboside 5'-diphosphate (thioGDP), from its complex with elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts). The mechanism of this reaction includes a ternary complex; EF-Tu.thioGDP.EF-Ts (Eccleston, J. F. (1984) J. Biol. Chem. 259, 12997-13003). This mechanism has been further investigated using pressure relaxation techniques combined with spectrophotometric measurements. The equilibrium of a solution of EF-Tu, EF-Ts, and thioGDP over a range of concentrations is perturbed on increasing the pressure to 150 atm. Rapid decrease of the pressure back to 1 atm results in a biphasic relaxation process, an initial fast phase which is complete within 1 ms followed by a slower phase. This is interpreted as the result of an isomerization of the EF-Tu.thioGDP.EF-Ts ternary complex which occurs before the release of thioGDP. Such an isomerization process may be a general feature in the release of GDP from guanosine nucleotide-binding proteins.