NMR study of isoleucine transfer RNA from Thermus thermophilus

An NMR and nuclear Overhauser effect (NOE) analysis of Thermus thermophilus tRNAIle1a is presented. This species contains modifications including s2T54 and s4U8 [Horie, N., Hara-Yokoyama, M., Yokoyama, S., Watanabe, K., Kuchino, Y., Nishimura, S., & Miyazawa, T. (1985) Biochemistry 24, 5711-5715]. All the expected secondary and reverse Hoogsteen AU pairs were identified, with one possible exception. The general geometry of the T psi C loop is the same as the Escherichia coli species, and there is NOE evidence for an A9-UA12 triple. Preliminary measurements of solvent exchange rates of internally hydrogen-bonded bases suggest that this tRNA is more stable than previously studied E. coli and yeast tRNAs.     

NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.

1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.     

Nuclear magnetic resonance observation and dynamics of specific amide protons in T4 lysozyme

We have produced T4 lysozyme using a bacterial expression system which allows efficient incorporation of isotopically labeled amino acids in lysozyme. By using conditions that repress the expression of various transaminases, we have incorporated 15N-labeled amino acid into the five phenylalanine residues of the protein. The relatively large spin--spin coupling (87 +/- 3 Hz) between the 15N nucleus and the phenylalanine amide protons may then be exploited in a variety of ways to selectively observe the five phenylalanine amide proton resonances. These include a simple "echo difference" technique which displays the amide proton resonances in one dimension and a "forbidden echo" technique [Bax, A., Griffey, R. H., & Hawkins, B.L. (1983) J. Magn. Reson. 55, 301-335] which gives two-dimensional information allowing the proton and 15N chemical shifts of each amide to be determined. With these approaches, all five phenylalanine amide protons give resolved resonances. Deuterium exchange experiments demonstrate that three of the five resonances are slow to exchange (half-times of about 1 week at pH 5.5 and 4 degrees C) while the other two are rapid with complete exchange in hours or less. These observations correlate well with the secondary structure of the protein which shows three residues in alpha-helical regions and two residues in surface-exposed environments. This approach of isotopic substitution on nitrogen or carbon atoms is of general utility and should allow virtually any proton on a protein of molecular weight 20 000 or thereabout to be selectively observed.     

Assignment of imino proton spectra of yeast phenylalanine transfer ribonucleic acid

Yeast tRNAPhe has been studied by using proton NMR and nuclear Overhauser effect (NOE) with deuterium substitution. Direct NOE evidence is presented for assignment of imino resonances of 23 of 27 base pairs in this tRNA. Other indirect evidence is presented for tentative assignment of four other base pairs. Almost total assignment also has been made of the important noninternally bonded imino protons and tertiary interactions (however, G18-psi 55 remains unassigned). The most surprising result has been identification of GC11 at -13.68 ppm; this is the first time a GC base pair has been identified so far downfield. This peak (GC11) is also identified as the resonance of the unique imino proton that exchanges in a time of more than 1 day, as previously described. These identifications of imino proton resonances made it possible to reinterpret the proton solvent exchange rate data previously published on this tRNA and understand them better. The assignments of resonances should pave the way for more detailed solution study of this tRNA and its interaction with biologically relevant molecules.     

The Haemophilus influenzae sxy-1 mutation is in a newly identified gene essential for competence.

The sxy-1 mutation of Haemophilus influenzae causes a 100- to 1,000-fold increase in spontaneous natural competence. We have used mapping and sequencing to identify this mutation as a G-to-A transition in an open reading frame adjacent to the rec-1 locus. This mutation substitutes valine for isoleucine at amino acid 19 of the protein specified by this gene (now named sxy). A multicopy plasmid containing the wild-type sxy gene confers constitutive competence on wild-type cells. Cells carrying this plasmid exhibit, in all stages of growth, DNA uptake levels and transformation frequencies as high those normally seen only after full induction of competence by starvation; deletion of part of the sxy gene from the plasmid abolishes this effect. In contrast, a transposon insertion in sxy entirely prevents both DNA uptake and transformation, indicating that sxy encodes a function essential for competence. These findings suggest that sxy may act as a positive regulator of competence. However, because cells carrying the transposon-inactivated sxy::Tn allele grow slowly under conditions that do not induce competence, sxy may also have a role in noncompetent cells.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Nuclear overhauser effect study of yeast aspartate transfer ribonucleic acid

Nuclear Overhauser effect studies are described for yeast tRNAAsp in 0.1 M NaCl, pH 7.0. A primary aim is to develop a general method for attacking the problem of assignment in transfer ribonucleic acids (tRNAs). Previously, we have demonstrated the utility of the nuclear Overhauser effect (NOE) between protons on adjacent base pairs combined with C8 deuterium substitution, by assigning the imino protons of the dihydrouridine stem and the two reverse-Hoogsteen base pairs T54-A58 and U8-A14. Here, we extend that approach to other parts of the molecule. We also describe several NOE-connected patterns for, e:g., m5CG and psi 55 N3H imino protons which may be of general utility. For the first time, a purine-15-pyrimidine-48 base pair (in this case A15-U48) has been assigned. A total of 13 of 25 base pairs from all parts of the molecule and several noninternally bonded imino protons have now been assigned unambiguously. This is a general method for assigning resonances in tRNA and perhaps in all double-stranded nucleic acids. This, and the distance information inherent in NOE measurements, should make NMR more generally applicable to nucleic acids.     

sxy-1, a Haemophilus influenzae mutation causing greatly enhanced spontaneous competence.

A Haemophilus influenzae strain carrying a competence-enhancing mutation (sxy-1) was selected by transformation of a mutagenized culture in exponential growth at low cell density, where spontaneous competence is very rare. Under these conditions, sxy-1 cells spontaneously transformed 100 to 1,000 times more efficiently than wild-type cells. Moreover, sxy-1 cells responded to all known competence-inducing treatments with further increases in transformation frequency. At high cell densities, sxy-1 cells spontaneously developed the level of competence reached by wild-type cells only after maximal induction by transfer to starvation medium. The sxy-1 mutation appears to act early in the sequence of events leading to competence; it increased the competence of cells carrying the early-acting transformation-defective (Tfo-) mutation tfo-98 by as large a factor as it did the competence of wild-type cells, but it had no effect when combined with another early-acting Tfo- mutation (tfo-87) or with the late-acting Tfo- mutation rec-2.