Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA

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.     

RfaH and the ops element, components of a novel system controlling bacterial transcription elongation

PURPOSE/OBJECTIVES: To describe the foundational work and implementation of a nurse practitioner (NP) curriculum geared toward oncology nurses. The study is selective (not comprehensive) and reflective of one school's experience. DATA SOURCE: Journal articles, curriculum guidelines, anecdotal experience, and interviews. DATA SYNTHESIS: The NP is used more frequently in oncology, both as a clinician and for other aspects of advanced practice nursing. NPs must be prepared to fulfill graduate criteria as outlined by definitive sources for curriculum development. CONCLUSIONS: Schools must work with employers, graduates, and patients in conducting outcome evaluations to measure safety issues and role effectiveness of oncology NPs (ONPs), as well as fulfillment of all aspects of the advanced nursing practice role. IMPLICATIONS FOR NURSING PRACTICE: If healthcare employers continue to rely heavily on the use of ONPs, schools of nursing must be prepared to graduate safe clinicians, experts in oncology, and advanced practice nurses, all combined into one graduate. This difficult task requires evaluation of current practices.     

Cell elongation and septation are two mutually exclusive processes in Escherichia coli

Bacterial rod morphogenesis was studied in synchronously growing cells of Escherichia coli C600 during the reshaping process that follows the removal of mecillinam, a beta-lactam antibiotic that specifically inhibits lateral wall formation of gram-negative rods and causes transition to coccal shape. Removal of mecillinam after 30 min of action did not affect the timing of subsequent cell division, but removal after 50 min delayed resumption of cell division for approximately one generation time. In order to study the interplay between lateral wall elongation and septum formation in determining and maintaining the bacterial rod shape, we evaluated the effect of re-adding mecillinam or of adding aztreonam (a specific inhibitor of septum formation) at various stages of the reshaping process. We conclude that mecillinam was active only during the reshaping process, while aztreonam was active only later when the cells were close to dividing again. These results provide further evidence for our previous proposal according to which elongation and septation are two alternating and competing events of the cell cycle and are linked to each other to force bacterial rods to grow to a given length.     

A new factor from Escherichia coli affects translocation of mRNA

Reconstitution of protein synthesis from purified translation factors on ribosomes from Escherichia coli has revealed the requirement for a protein, W, that affects chain elongation and is essential to reconstitute the process (Ganoza, M. C., Cunningham, C., and Green, R. M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1648-1652). We report that W has no effect on initiation complex formation by 30 or 70 S ribosomes or on the association of ribosomal subunits, peptide bond synthesis, or binding Ala-tRNA, which is the second amino acid of the coat protein of the MS2 RNA virion. W has a pronounced effect on tripeptide synthesis, and is obligatory for the synthesis of the coat protein or of the hexapeptide encoded by f2am3 RNA. Extracts from a temperature-sensitive mutant of the translocase, EF-G, were purified free of the W protein and were used to score for translocation defects. W is required for binding Ser-tRNA, the third N-terminal amino acid of the MS2 or f2 RNA coat protein to ribosomes bearing fMet-Ala-tRNA, as well as for the ejection of deacyl-tRNA from ribosomes, which occurred concomitant with the binding of the Ser-tRNA. We propose that W functions by ejecting tRNAs from ribosomes in a step that precedes the movement of mRNA during translocation.     

Role of domains in Escherichia coli and mammalian mitochondrial elongation factor Ts in the interaction with elongation factor Tu

Bovine mitochondrial elongation factor Ts (EF-Tsmt) stimulates the activity of Escherichia coli elongation factor Tu (EF-Tu). In contrast, E. coli EF-Ts is unable to stimulate mitochondrial EF-Tu. EF-Tsmt forms a tight complex with E. coli EF-Tu governed by an association constant of 8.6 x 10(10). This value is 100-fold stronger than the binding constant for the formation of the E. coli EF-Tu.Ts complex. To test which domain of EF-Tsmt is important for its strong binding with EF-Tu, chimeras were made between E. coli EF-Ts and EF-Tsmt. Replacing the N-terminal domain of E. coli EF-Ts with that of EF-Tsmt increases its binding to E. coli EF-Tu 2-3-fold. Replacing the N-terminal domain of EF-Tsmt with the corresponding region of E. coli EF-Ts decreases its binding to E. coli EF-Tu approximately 4-5-fold. A chimera consisting of the C-terminal half of E. coli EF-Ts and the N-terminal half of EF-Tsmt binds to E. coli EF-Tu as strongly as EF-Tsmt. A chimera in which Subdomain N of the core of EF-Ts is replaced by the corresponding region of EF-Tsmt binds E. coli EF-Tu approximately 25-fold more tightly than E. coli EF-Ts. Thus, the higher strength of the interaction between EF-Tsmt and EF-Tu can be localized primarily to a single subdomain.     

Mutational analysis of the roles of residues in Escherichia coli elongation factor Ts in the interaction with elongation factor Tu

The crystal structure of the Escherichia coli elongation factor (EF)-Tu.Ts complex indicates that there are extensive contacts between EF-Tu and EF-Ts. To determine the importance of these contacts in the interaction between E. coli EF-Tu and EF-Ts, residues in EF-Ts at the interface of these two proteins were mutated. The binding constants governing the interaction of the resulting EF-Ts variants with E. coli EF-Tu were determined. The effects of these mutations on the ability of EF-Ts to stimulate GDP exchange with EF-Tu.GDP and on its ability to stimulate the activity of EF-Tu in polymerization were tested. The results indicate that Arg-12, Met-19, and Met-20 in the N-terminal domain of EF-Ts and His-147 and Lys-166 and/or His-167 in subdomain C of EF-Ts are crucial in the interaction between EF-Tu and EF-Ts. Lys-23, Val-234, Met-235, and the C-terminal helix h13 are less important. The binding constants of the EF-Ts variants governing their interactions with EF-Tu correlate well with their activities in stimulating GDP exchange with EF-Tu. Mutations prepared in EF-Tu indicate that His-19 and Gln-114 but not Glu-348 in EF-Tu are moderately important for its interaction with EF-Ts.     

Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli

1. The effect of increasing cellular tyrosine phosphorylation by inhibiting endogenous tyrosine phosphatases was examined on voltage-operated calcium channel currents in vascular smooth muscle cells. 2. In single ear artery smooth muscle cells of the rabbit, studied by the whole cell voltage clamp technique, intracellular application of the tyrosine phosphatase inhibitors, sodium orthovanadate (100 microM) and peroxyvanadate (100 microM orthovanadate + 1 mM H2O2) increased voltage-operated calcium channel currents by 56% and 83%, respectively. 3. Bath application of two other membrane permeant tyrosine phosphatase inhibitors, phenylarsine oxide (100 microM) and dephostatin (50 microM) also increased voltage-operated calcium channel currents by 48% and 52%, respectively. 4. The selective tyrosine kinase inhibitor, tyrphostin-23 (100 microM) reduced calcium channel currents by 41%. Pre-incubation with tyrphostin-23 abolished the effects of peroxyvanadate, phenylarsine oxide and dephostatin on calcium channels. 5. Western blot analysis of rabbit ear artery cell lysates showed increased tyrosine phosphorylation of several endogenous proteins following treatment with peroxyvanadate. 6. These results indicate that a number of structurally dissimilar inhibitors of tyrosine phosphatases increase voltage-operated calcium channel currents in arterial smooth muscle cells presumably due to increased tyrosine phosphorylation.     

Identification of protein synthesis elongation factor G as a 4.5 S RNA-binding protein in Escherichia coli

Escherichia coli 4.5 S RNA is metabolically stable and abundant. It consists of 114 nucleotides, and it is structurally homologous to domain IV of mammalian signal recognition particle (SRP) RNA. In this study, we found two 4.5 S RNA-binding proteins in cell extracts by means of a gel mobility shift assay. One protein was identified as Ffh, which has been characterized as 4.5 S RNA-binding protein. The other protein was separated from Ffh by two consecutive column chromatographic elutions and by monitoring the 4.5 S RNA binding activity. After the second chromatography, a dominant protein with an approximate molecular weight of 78,000 was associated with 4.5 S RNA binding activity. A sequence of the NH2-terminal 19 residues of the 78-kDa protein was completely identical to that of the protein elongation factor G (EF-G) of E. coli, and further it cross-reacted with antiserum against E. coli EF-G. The results obtained using a synthetic oligo RNA corresponding to the 23 S rRNA defining the EF-G binding site indicated that 4.5 S RNA and 23 S rRNA are competitive in 4.5 S RNA binding and that a decanucleotide sequence conserved between them serves as a binding site for EF-G. Conservation of the SRP RNA binding activity of EF-G from Bacillus subtilis suggests that the binding of EF-G to SRP RNA is essential for its function.     

Visualization of elongation factor G on the Escherichia coli 70S ribosome: the mechanism of translocation

During protein synthesis, elongation factor G (EF-G) binds to the ribosome and promotes the step of translocation, a process in which tRNA moves from the A to the P site of the ribosome and the mRNA is advanced by one codon. By using three-dimensional cryo-electron microscopy, we have visualized EF-G in a ribosome-EF-G-GDP-fusidic acid complex. Fitting the crystal structure of EF-G-GDP into the cryo density map reveals a large conformational change mainly associated with domain IV, the domain that mimics the shape of the anticodon arm of the tRNA in the structurally homologous ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. The tip portion of this domain is found in a position that overlaps the anticodon arm of the A-site tRNA, whose position in the ribosome is known from a study of the pretranslocational complex, implying that EF-G displaces the A-site tRNA to the P site by physical interaction with the anticodon arm.     

Crystal structure of intact elongation factor EF-Tu from Escherichia coli in GDP conformation at 2.05 A resolution

The crystal structure of intact elongation factor Tu (EF-Tu) from Escherichia coli in GDP-bound conformation has been determined using a combination of multiple isomorphous replacement (MIR) and multiwavelength anomalous diffraction (MAD) methods. The current atomic model has been refined to a crystallographic R factor of 20.3 % and free R-factor of 26.8 % in the resolution range of 10-2.05 A. The protein consists of three domains: domain 1 has an alpha/beta structure; while domain 2 and domain 3 are beta-barrel structures. Although the global fold of the current model is similar to those of published structures, the secondary structural assignment has been improved due to the high quality of the current model. The switch I region (residues 40-62) is well ordered in this structure. Comparison with the structure of EF-Tu in GDP-bound form from Thermus aquaticus shows that although the individual domain structures are similar in these two structures, the orientation of domains changes significantly. Interactions between domains 1 and 3 in our E. coli EF-Tu-GDP complex are quite different from those of EF-Tu with bound GTP from T. aquaticus, due to the domain rearrangement upon GTP binding. The binding sites of the Mg2+ and guanine nucleotide are revealed in detail. Two water molecules that co-ordinate the Mg2+ have been identified to be well conserved in the GDP and GTP-bound forms of EF-Tu structures, as well as in the structure of Ras p21 with bound GDP. Comparisons of the Mg2+ binding site with other guanine nucleotide binding proteins in GDP-bound forms show that the Mg2+ co-ordination patterns are well preserved among these structures.     

Polyadenylation promotes degradation of 3'-structured RNA by the Escherichia coli mRNA degradosome in vitro

Polyadenylation contributes to the destabilization of bacterial mRNA. We have investigated the role of polyadenylation in the degradation of RNA by the purified Escherichia coli degradosome in vitro. RNA molecules with 3'-ends incorporated into a stable stem-loop structure could not readily be degraded by purified polynucleotide phosphorylase or by the degradosome, even though the degradosome contains active RhlB helicase which normally facilitates degradation of structured RNA. The exoribonucleolytic activity of the degradosome was due to polynucleotide phosphorylase, rather than the recently reported exonucleolytic activity exhibited by a purified fragment of RNase E (Huang, H., Liao, J., and Cohen, S. N. (1998) Nature 391, 99-102). Addition of a 3'-poly(A) tail stimulated degradation by the degradosome. As few as 5 adenosine residues were sufficient to achieve this stimulation, and generic sequences were equally effective. The data show that the degradosome requires a single-stranded "toehold" 3' to a secondary structure to recognize and degrade the RNA molecule efficiently; polyadenylation can provide this single-stranded 3'-end. Significantly, oligo(G) and oligo(U) tails were unable to stimulate degradation; for oligo(G), at least, this is probably due to the formation of a G quartet structure which makes the 3'-end inaccessible. The inaccessibility of 3'-oligo(U) sequences is likely to have a role in stabilization of RNA molecules generated by Rho-independent terminators.     

Diarrheagenic Escherichia coli

Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler's diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (entero-pathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens.     

The "allosteric three-site model" of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli

For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed. It has been suggested that transient E-site binding of the tRNA leaving the peptidyl (P) site promotes elongation factor G (EF-G)-dependent translocation by lowering the energetic barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W. (1989) EMBO J. 8, 3933-3938]. The alternative "allosteric three-site model" [Nierhaus, K.H. (1990) Biochemistry 29, 4997-5008] features stable, codon-dependent tRNA binding to the E site and postulates a coupling between E and aminoacyl (A) sites that regulates the tRNA binding affinity of the two sites in an anticooperative manner. Extending our testing of the two conflicting models, we have performed translocation experiments with fully active ribosomes programmed with heteropolymeric mRNA. The results confirm that the deacylated tRNA released from the P site is bound to the E site in a kinetically labile fashion, and that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ or polyamines. At conditions of high E-site occupancy in the posttranslocation complex, filling the A site with aminoacyl-tRNA had no influence on the E site, i.e., there was no detectable anticooperative coupling between the two sites, provided that second-round translocation was avoided by removing EF-G. On the basis of these results, which are entirely consistent with our previous results, we consider the allosteric three-site model of elongation untenable. Rather, as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.     

Escherichia coli diarrhea

E. coli diarrheal disease is becoming ever more complicated as more and more pathogenic mechanisms are identified. E. coli strains remain the major causes of infectious diarrhea worldwide. Presumptive diagnosis based on clinical and laboratory criteria is practical for strains known to be important in the United States. Specific diagnosis is not currently feasible outside of research centers. Therapy, when indicated, shortens the duration of illness. Research is proceeding rapidly at the molecular level and may lead to new diagnostic and therapeutic approaches in the near future.     

Granulocyte colony-stimulating factor enhances protection by anti-K1 capsular IgM antibody in murine Escherichia coli sepsis

Combined prophylactic treatment with recombinant murine granulocyte colony-stimulating factor (G-CSF) and a suboptimal dose of anti-K1 capsular IgM monoclonal antibody (MAb) significantly enhanced survival in an experimental mouse Escherichia coli O7:K1 peritonitis model compared with untreated animals (67% vs. 11% survival; P < 0.001) and with either treatment alone (67 vs. 29% and 27% survival, respectively; P < 0.01), which suggests synergism between these agents. Enhanced survival by combined treatment was associated with increased neutrophil counts in blood and peritoneal lavage fluid, lower systemic and higher levels of local tumour necrosis factor (TNF) and lower bacterial counts in blood cultures. Mouse neutrophils treated with G-CSF but not infected with E. coli showed enhanced phagocytic and respiratory burst capacity, down-regulation of L-selectin receptors and enhanced expression of Fc RII-III receptors but not of complement receptors.