Early evolutionary relationships among known life forms inferred from elongation factor EF-2/EF-G sequences: Phylogenetic coherence and structure of the archaeal domain

Cammarano, Piero; Palm, Peter; Creti, Roberta; Ceccarelli, Elena; Sanangelantoni, Anna Maria; Tiboni, Orsola

doi:10.1007/bf00162996

Cited by 58 publications

(24 citation statements)

References 51 publications

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“…This result is consistent with the previous observation that B. subtilis EF-G binds to 4.5S RNA from both E. coli and Clostridium perfringens (Shibata et al 1996). Interestingly, the sequence conservation between the archaeal and bacterial proteins is only ∼30%, with the highest conservation in the G domain responsible for GTP binding and hydrolysis (Cammarano et al 1992;Creti et al 1994;Thomas and Cavicchioli 1998). In contrast, the sequence of domain IV of 4.5S RNA is highly conserved in all three kingdoms of life, supporting the idea that this part of the RNA arose early in evolution, and that its sequence is constrained by multiple interacting factors.…”

Section: Discussionsupporting

confidence: 92%

Conserved but nonessential interaction of SRP RNA with translation factor EF-G

Sagar

Lucast²,

Doudna³

2004

RNA

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ABSTRACT4.5S RNA is essential for viability of Escherichia coli, and forms a key component of the signal recognition particle (SRP), a ubiquitous ribonucleoprotein complex responsible for cotranslational targeting of secretory proteins. 4.5S RNA also binds independently to elongation factor G (EF-G), a five-domain GTPase that catalyzes the translocation step during protein biosynthesis on the ribosome. Point mutations in EF-G suppress deleterious effects of 4.5S RNA depletion, as do mutations in the EF-G binding site within ribosomal RNA, suggesting that 4.5S RNA might play a critical role in ribosome function in addition to its role in SRP. Here we show that 4.5S RNA and EF-G form a phylogenetically conserved, low-affinity but highly specific complex involving sequence elements required for 4.5S binding to its cognate SRP protein, Ffh. Mutational analysis indicates that the same molecular structure of 4.5S RNA is recognized in each case. Surprisingly, however, the suppressor mutant forms of EF-G bind very weakly or undetectably to 4.5S RNA, implying that cells can survive 4.5S RNA depletion by decreasing the affinity between 4.5S RNA and the translational machinery. These data suggest that SRP function is the essential role of 4.5S RNA in bacteria.

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Section: Discussionsupporting

confidence: 92%

Conserved but nonessential interaction of SRP RNA with translation factor EF-G

Sagar

Lucast²,

Doudna³

2004

RNA

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“…These mutations were directly adjacent to conserved sequences (found near the carboxyl terminus of the protein; Fig. 6) that occur in all ribosome translocases sequenced to date, both prokaryotic and eukaryotic (5,14).…”

Section: Resultsmentioning

confidence: 99%

“…initially described amino acid sequences found in the carboxylterminal portion of eukaryotic EF-2 which were similar to sequences found in the corresponding region of E. coli EF-G (14). A more complete comparison of EF-G and EF-2 sequences representing eubacteria, archaebacteria, and eukaryotes has recently been presented (5). Inspection of these data indicate that many of the carboxyl-terminal positions originally discussed by Kohno et al (14) appear to be well conserved.…”

Section: Discussionmentioning

confidence: 95%

“…These missense mutations are both directly adjacent to a block of amino acid residues that are conserved in all bacterial EF-G genes that have been sequenced as well as all eukaryotic EF-2-encoding genes. It is concluded that this region of the protein is required for an EF-G function that is ancient since it is encoded by sequences found in organisms as diverse as rodents and bacteria (5,14).…”

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confidence: 99%

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In vivo selection of conditional-lethal mutations in the gene encoding elongation factor G of Escherichia coli

et al. 1994

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The ribosome translocation step that occurs during protein synthesis is a highly conserved, essential activity of all cells. The precise movement of one codon that occurs following peptide bond formation is regulated by elongation factor G (EF-G) in eubacteria or elongation factor 2 (EF-2) in eukaryotes. To begin to understand molecular interactions that regulate this process, a genetic selection was developed with the aim of obtaining conditional-lethal alleles of the gene (fussA) that encodes EF-G in Escherichia coli. The genetic selection depends on the observation that resistant strains arose spontaneously in the presence of sublethal concentrations of the antibiotic kanamycin. Replica plating was performed to obtain mutant isolates from this collection that were restrictive for growth at 42°C. Two tightly temperature-sensitive strains were characterized in detail and shown to harbor single-site missense mutations within Jis4. ThefusA4100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fus4101 allele encoded a glutamine-to-proline alteration at position 495. Induction kinetics of 0-galactosidase activity suggested that both mutations resulted in slower elongation rates in vivo.These missense mutations were very near a small group of conserved amino acid residues (positions 483 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded that these sequences encode a specific domain that is essential for efficient translocase function.In bacteria, the elongation steps of protein synthesis require the sequential action of two different elongation factors, EF-G and EF-Tu. EF-Tu is required to deliver the correct aminoacyltRNA to the A site on the ribosome and is, therefore, intimately involved in proofreading (for a review, see reference 27). EF-G catalyzes ribosome translocation on mRNA through a mechanism that somehow is enhanced by hydrolysis of GTP by EF-G (for a review, see reference 15). The movement of the ribosome is an intricate mechanical process that remains relatively poorly understood. Previous biochemical investigations have been aimed at defining the sites at which EF-G binds to ribosomal protein and rRNA. From these data, it is apparent that EF-G binds to the ribosome at the base of the L7/L12 stalk and is closely associated with proteins L10 and Lii among others (for a review, see reference 26). Crosslinking and RNA protection experiments provide convincing evidence that part of the binding site is also composed of the 23S rRNA near nucleotides 2660 and 1067 (21, 24). In addition, in vitro experiments have shoWn that the L7/L12 protein dimer is essential for EF-G binding and function (10,13,22). Little is known about the residues in EF-G which participate in these interactions. In particular, there have been relatively few investigations that use bacterial genetics to select for informative mutations in fusA (the Escherichia coli gene that encodes EF-G). One of the early studies used the incorporation of tritiated precursors to induce death by suicide (25). A temperatur...

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“…As an additional tool for the classification of phytoplasmas, the conserved gene encoding the elongation factor TU (tuf gene), which has been widely used as a phylogenetic marker (Sela et al, 1989 ;Cammarano et al, 1992 ;Ludwig et al, 1993 ;Ceccarelli et al, 1995 ;Kamla et al, 1996), was examined by Schneider et al (1997a). By RFLP analysis of the PCR-amplified tuf gene using Sau3AI restriction endonuclease, 21 AY isolates could be divided into five subgroups.…”

Section: Introductionmentioning

confidence: 99%