Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo

Abstract

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.The small basic protein cr-sarcin has been shown to inhibit protein synthesis in Escherichia coli 111 by specifically cleaving the phosphodiester bond on the 3'-side of the G2661 residue. This base is located in the so called cr-sarcin loop in domain six of the 23s rRNA [2]. Since the cleavage blocks the binding of both EF-Tu . GTP . aminoacyl-tRNA and EF-G . GTP to the ribosome, the G2661 residue is presumably located at the overlapping region of the binding sites of both elongation factors 121.Recently, a single base substitution mutation was constructed in the cloned rrnB operon of the multi-copy plasmid pKK3535, altering the base at position 2661 in 23s rRNA from G to C [3]. The translational elongation rate of the mutant 2661 C ribosomes, measured in a poly(U)-primed in vitro system, was lowered if the ribosomes also were altered in ribosomal protein S12. This effect was suggested to be the result of stronger binding of EF-Tu . GTP . aminoacyl-tRNA ternary complexes to mutant ribosomes [3].Many streptomycin-resistant mutations in the rpsL gene, encoding ribosomal protein S12, are known to increase the accuracy of translation [4, 51. One such example, giving a hyper-accurate (restrictive) phenotype, is the rpsL282 mutation. In contrast, another mutation in this gene, rpsL224, leads to streptomycin resistance although it still shows an essentially ...

Structural Insights Into Translational Fidelity

“…Further, the G2661C/S12-SmP double mutant can be rescued not only by Smmediated reinforcement at the shoulder, but also by certain kirromycin-resistant (KirrR) mutants of EF-Tu (173). These KirrR mutations of EF-Tu alone ( Figure 5) cause an error-prone phenotype.…”

Evidence for functional interaction between elongation factor Tu and 16S ribosomal RNA.

“…Recently, a G --C mutation at position 2661 in the a-sarcin loop has been shown to impair translational elongation, but, surprisingly, only in combination with restrictive streptomycin-resistance mutations in protein S12 (40). The functional impairment of this double mutant is suppressed by a third mutation, in EF-Tu itself (41). Furthermore, it has been observed that the normally recessive kirromycin resistance caused by mutations in EF-Tu becomes dominant in certain restrictive S12 mutant backgrounds (42).…”

Probing Surface Structure of Sarcin Domain on Ribosomes of <i>Escherichia coli</i> by Complementary Oligo DNAs and Ribosome-Inactivating Protein

“…The nucleotides at the sarcin domain are involved in elongation factors catalyzing the binding of aminoacyltRNA to ribosomes [34]. These nucleotides may be involved in factors catalyzing the translocation, because treatment of ·-sarcin aborts these reactions [28,34].…”

“…These nucleotides may be involved in factors catalyzing the translocation, because treatment of ·-sarcin aborts these reactions [28,34]. Aminoacyl-tRNA, peptidyl-tRNA, or both have been shown to be bound to the ribosome and to protect against the treatment of ·-sarcin [22,25,30].…”

“…The loop is the longest known universally conserved sequence of rRNA [12,38]. Changes of certain nucleotides on the loop would greatly affect the growth rate, ribosome assembly and entry of mutant ribosomes into polysomes [18,34], and its reactivity to ribosome-inactivating protein [8,31,33].…”