An A to U transversion at position 1067 of 23 S rRNA from Escherichia coli impairs EF-Tu and EF-G function 1 1 Edited by D. E. Draper (original) (raw)
Related papers
Journal of Molecular Biology, 1997
Escherichia coli ribosomes with an A to U transversion at nucleotide 1067 of their 23 S rRNA are impaired in their effective association rate constants (k cat /K M ) for both EF-Tu and EF-G binding. In addition, the times that EF-G and EF-Tu spend on the ribosome during elongation are sig-ni®cantly increased by the A to U transversion. The U1067 mutation impairs EF-Tu function more than EF-G function. The increase in the time that EF-Tu remains bound to ribosome is caused, both by a slower rate of GTP-hydrolysis in ternary complex and by a slower EF-Tu Á GDP release from the mutated ribosomes. There is, at the same time, no change in ribosomal accuracy for aminoacyl-tRNA recognition. With support from these new data we propose that nucleotide 1067 is part of the ribosomal A-site where it directly interacts with both EF-G and EF-Tu.
European Journal of Biochemistry, 1991
The binding of the EF-Tu . GTP . aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23s ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23s rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as norinal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661 C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wildtype form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2662C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661 C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2662C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23s rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.
Elongation factor Tu (EF-Tu) GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tufgene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EF-Tu. GTP aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed. chem. 75:67-75.
Function of the ribosomal E-site: a mutagenesis study
Nucleic Acids Research, 2005
Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.
Mapping Escherichia coli Elongation Factor Tu Residues Involved in Binding of Aminoacyl-tRNA
Journal of Biological Chemistry, 1996
Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl-tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay. Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA. Elongation factor Tu (EF-Tu) 1 is a key enzyme in protein biosynthesis. In its active conformation with GTP as a cofactor, it carries aminoacyl-tRNA (aa-tRNA) to the A-site of the programmed ribosome in the process of decoding mRNA (1). The complex of EF-Tu, GTP, and aa-tRNA has been denoted the ternary complex. During peptide bond formation GTP is hydrolyzed and EF-Tu adopts its inactive conformation, which has a negligible affinity for aa-tRNA. Nucleotide exchange factor EF-Ts catalyzes the replacement of GDP with GTP thereby completing the cycle. EF-Tu-dependent GTP hydrolysis is a rate-limiting step in protein biosynthesis and consequently is considered to be a step for proofreading of mRNA. The ribosome and aa-tRNA act as stimulating effectors of the GTPase activity at this crucial step. In Escherichia coli peptide bond formation proceeds at a rate of 10-20 bonds/s/ribosome (2). E. coli EF-Tu is a monomeric protein consisting of 393 amino acid residues and with a molecular mass of 43.2 kDa. The nucleotide sequences of the two EF-Tu encoding genes tufA and tufB have been determined (3, 4) and so has the amino acid * This work was funded by the Danish Biotechnology program (PERC) and the NOVO fund.
Topography of the E site on the Escherichia coli ribosome
The EMBO journal, 1993
Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes. Two of the probes were derivatives of E. coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine. The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine. Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E. coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits. The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit. The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucl...
Journal of Biological Chemistry, 1999
The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mM MgCl 2 , 0.5 M NH 4 Cl, at 30°C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.
Uniform Binding of Aminoacylated Transfer RNAs to the Ribosomal A and P Sites
Molecular Cell, 2004
a diverse set of substrates in an equivalent manner. and Cell Biology The majority of biochemical experiments exploring Northwestern University the function of tRNAs in translation have focused on a Evanston, Illinois 60208 single tRNA, Phe-tRNA Phe (Lill et al., 1986; Joseph et al., 1997; Yoshizawa et al., 1999; Wower et al., 2000, Ogle et al, 2001)