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B Kraal

Publications and source records attributed to B Kraal.

At least 55 records · Page 3Linked to original sources
PubMed1984

Cross-linking of tRNA at two different sites of the elongation factor Tu.

Recently, we reported on the induction by kirromycin of two tRNA binding sites on elongation factor Tu. To obtain independent information on the existence of these two sites and to characterize them further, 3' oxidized tRNA was cross-linked to elongation factor Tu by [3H]borohydride reduction. Specific cross-linking occurred exclusively in the presence of kirromycin. In the case of elongation factor Tu X GDP X kirromycin, cross-linking was found at lysine-208; in elongation factor Tu X GTP X kirromycin, cross-linking was at lysine-208 and lysine-237. In both elongation factor Tu complexes, kirromycin itself was found cross-linked to lysine-357. The tRNA cross-linking sites are in agreement with the idea of two different binding sites of tRNA on elongation factor Tu.

Anti-Bacterial Agents↗
PubMed1982

The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules.

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Bacterial Agents↗
PubMed1981

Molecular properties of two mutant species of the elongation factor Tu.

The molecular properties of two mutant species of the elongation factor Tu (EF-Tu), derived from either tuf A or tuf B, have been studied. One, designated EF-TuAR, is the product of a kirromycin-resistant tufA gene. The other designated EF-TuBO is a tuf B product and is present in a kirromycin-resistant mutant of Escherichia coli (LBE 2012) also harbouring the EF-TuAR species. EF-TuAR has been isolated in homogeneous form as a single gene product from the mutant strain LBE 2045, in which the tuf B gene has been inactivated by an insertion of the bacteriophage Mu. EF-TuBO has been isolated from LBE 2012 together with EF-TuAR in a 1:1 mixture. Fractionation of this mixture of DEAE-Sephadex A-50 resulted in an enrichment of EF-TuBO of about 80%. The properties of EF-TuAR and EF-TuBO have been compared to those of a kirromycin-sensitive species designated EF-TuAS, which was isolated from LBE 2045 by transduction of wild-type tuf A. We show here that all three EF-Tu species are fully competent to sustain polypeptide synthesis. All also appear to interact normally with guanine nucleotides and EF-Ts. Only in the presence of the antibiotic do the following differences appear. (a) Kirromycin causes EF-TuAS (wild-type tuf A gene product) to be retained on, and thus block, the ribosome. (b) EF-TuAR fails to bind the antibiotic and thus is capable of protein synthesis in its presence. (c) EF-TuBO fails to sustain polypeptide synthesis upon binding of kirromycin. It does not, however, block the ribosome, so the strain harbouring both this protein and EF-TuAR (LBE 2012) is kirromycin resistant.

Anti-Bacterial Agents↗
PubMed1981

A mutant elongation factor Tu which does not immobilize the ribosome upon binding of kirromycin.

In the accompanying paper we have shown that polypeptide synthesis sustained by the mutant elongation factor EF-TuBO is inhibited by kirromycin. Here we have searched for the primary site of inhibition in the elongation cycle. It is demonstrated that in the presence of the antibiotic EF-TuBO can form a complex with aminoacyl-tRNA and GTP and that the complex is able to bind to ribosomes programmed with poly(U). Like its wild-type counterpart, EF-TuBO . GDP can form a quaternary complex with aminoacyl-tRNA and kirromycin but, unlike the wild-type quaternary complex, the mutant complex fails to associate with the ribosome. This explains the recessive nature of the tuf B mutation in cells producing kirromycin-resistant EF-TuA and EF-TuBO. It also suggests a mechanism for the inhibition by kirromycin of EF-TuBO-dependent polypeptide synthesis.

Anti-Bacterial Agents↗
PubMed1979

Translation by Escherichia coli ribosomes of alfalfa mosaic virus RNA 4 can be initiated at two sites on the monocistronic message.

Substantial evidence is provided to corroborate our previous finding that Escherichia coli ribosomes recognize two binding sites on the 5' end of alfalfa mosaic virus (AMV) RNA 4 [for a preliminary report see Castel, A., Kraal, B., Kerklaan, P. R. M., Klok, J., and Bosch, L. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 5509--5513]. Translation can start at either site using AcPhe-tRNA or fMet-RNA as initiator and takes place in the same reading frame along the monocistronic mRNA. The size and composition of the isolated extra NH2-terminal fragment of the acetylphenylalanyl product were found to be in agreement with the 5' non-coding region of the messenger. Removal of the 5'-terminal cap structure of AMV RNA 4 did not influence significantly both initiation reactions. Ribosomal protein S1 was essential for binding as well as incorporation of both fMet-tRNA and AcPhe-tRNA. A similar interaction on the ribosome was found for AcPhe-tRNA directed by AMV RNA 4 as for fMet-tRNA directed by either AMV RNA 4 or MS2 RNA with respect to the influence of initiation factors. It is concluded that the heterologous plant viral messenger is reliably translated in the E. coli system and that E. coli ribosomes recognize with high specificity an extra initiation site close to the 5' extremity of the messenger. The relationship of this site to a hypothetical entry site involved in the early recognition in the initiation mechanism between ribosome and messenger is discussed.

Escherichia coli↗
PubMed1979

The primary structure of the coat protein of alfalfa mosaic virus strain VRU. A hypothesis on the occurrence of two conformations in the assembly of the protein shell.

The complete primary structure of the coat protein of strain VRU of alfalfa mosaic virus (AMV) is reported. The strain is morphologically different from all other AMV strains as it contains large amounts of unusually long virus particles. This is caused by structural differences in the coat protein chain. The amino acid sequence has mainly been established by the characterization of peptides obtained after cleavage with cyanogen bromide and digestion with trypsin, chymotrypsin, thermolysin or Staphylococcus aureus protease. The major sequencing technique used was the dansyl-Edman procedure. The VRU coat protein consists of 219 amino acid residues corresponding to a molecular weight of 24056. Compared to the coat protein of strain 425 [Van Beynum et al. (1977) Eur. J. Biochem. 72, 63-78], 15 amino acid substitutions were localized. Most of them have a conservative character and may be explained by single-point mutations. A correction is given for the AMV 425 coat protein: Asn-216 was shown to be Asp-216. The prediction of the secondary structure for the two viral coat proteins was not significantly influenced by the various amino acid substitutions except for the region containing residues 65-100. This led us to the hypothesis that the AMV coat protein may occur in two different conformations favouring its incorporation into either a pentagonal or hexagonal quasi-equivalent position in the lattice of the protein shell. The substitutions in the above-mentioned region of the VRU coat protein may have caused a strong preference for the hexagonal lattice conformation. The model is supported by preliminary sequence data of the same coat protein region in AMV 15/64, a strain morphologically intermediate between 425 and VRU.

Amino Acid Sequence↗
PubMed1977

Structural studies on the coat protein of alfalfa mosaic virus. The complete primary structure.

The complete amino acid sequence of the coat protein of alfalfa mosaic virus (strain 425) is reported. Sequence determinations were mainly performed on peptides obtained from fragmentation by cyanogen bromide and trypsin. Both manual and automatic sequence methods were used. Some refinements of the solid-phase Edman degradation were introduced. The final alignment of the peptides was established by means of alternative cleavage methods, such as limited tryptic digestion of intact virus particles, tryptic digestion after blockage of lysine residues and chymotryptic digestion. The coat protein consists of 220 amino acid residues corresponding to a molecular weight of 24252. A remarkable clustering of basic residues occurs in the N-terminal part of the protein chain. Several internal hydrophobic clusters and a strongly acidic site at the C-terminus can be observed. Two regions of sequence homology (12 residues) were found. Some features of the secondary structure are predicted.

Amides↗
PubMed1977

Initiation of polypeptide synthesis with various NH2-blocked aminoacyl-tRNAs under the direction of alfalfa mosaic virus RNA 4.

Initiation of polypeptide synthesis in a cell-free system of Escherichia coli directed by alfalfa mosaic virus RNA 4 was studied by using either fMet-tRNA or Ac-Phe-tRNA as initiator tRNA. Initiation with fMet-tRNA yielded a product that was identical to the authentic viral coat protein except that the NH2-terminal serine was preceded by fMet instead of being acetylated. When Ac-Phe-tRNA was used as initiator, the biosynthetic product was 10-12 amino acid residues longer, the extra amino acids being located at the NH2-terminus. fMet-tRNA and Ac-Phe-tRNA did not compete for ribosomes during initiation of protein synthesis, as became evident from incorporation studies using both initiator tRNAs simultaneously. It is concluded that E. coli ribosomes recognize two sites on the 5' end of alfalfa mosaic virus RNA 4 that are separated by a region of about 30 nucleotides. The results are in complete agreement with the 5'-terminal nucleotide sequence of this RNA [Koper-Zwarthoff, E. C., Lockhard, R. E., RajBhandary, U. L., Alzner-deWeerd, B. & Bol, J. F. (1977) Proc. Natl. Acad. Sci. USA 74, 5504-5508].

Cell-Free System↗
PubMed1975

Structural studies on the coat protein of alfalfa mosaic virus. Isolation and characterization of the tryptic peptides and the alignment of the cyanogen-bromide fragments.

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen-bromide cleavage. The tryptic peptides from the protein and its four cyanogen-bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end-group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.

Amino Acid Sequence↗