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B R Reid

Publications and source records attributed to B R Reid.

At least 91 records · Page 5Linked to original sources
PubMed1979

Nuclear magnetic resonance studies on transfer ribonucleic acid: assignment of AU tertiary resonances.

The hydrogen-bonded ring NH nuclear magnetic resonance (NMR) spectra of several transfer ribonucleic acid (RNA) species have been examined with particular emphasis on the extreme low-field portion. Betwen --13.8 and --15 ppm there are two extra resonances which are not derived from cloverleaf base pairs. A combined approach involving undermodified tRNAs, chemical modification, and hairpin fragment studies has assigned the T54--A58 resonance at --14.3 ppm in yeast tRNAPhe and Escherichia coli tRNA1 Val., the U8--A14 resonance has been assigned at --14.3 ppm, and the s4U8--A14 resonance in bacterial tRNAs has been assigned at --14.9 ppm. The T54--A58 resonance shifts between --14.3. and --13.8 ppm depending on the surrounding nucleotide sequence in the ribothymidine loop.

Base Composition↗
PubMed1979

Paramagnetic ion effects on the nuclear magnetic resonance spectrum of transfer ribonucleic acid: assignment of the 15--48 tertiary resonance.

We have studied the effects of Co2+ and Mn2+ ions on the low-field nuclear magnetic resonance (NMR) spectra of pure class 1 transfer ribonucleic acid (tRNA) species. With 1.2 mM tRNA in the presence of 15 mM MgCl2 discrete paramagnetic effects were observed for Co2+ at concentrations in the range 0.02--0.1 mM and for Mn2+ in the range 0.002--0.01 mM, indicating fast exchange of these cations with tRNA. Both of these cations paramagnetically relax the s4U8--A14 resonance as well as other resonances from proximal base pairs. The Co2+ site appears to be the same site on G15 which was observed crystallographically [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315-328]; the initially occupied tight Mn2+ site is the cation site involving the phosphate of U8. There are three base pairs within 10 A of both sites, namely, G15--C48, A14--s4U8, and C13--G22; this has led to the assignment of the G15--C48 and C13--G22 resonances in the NMR spectrum [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315--328; Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, Sung-Hou (1977) Nucleic Acids Res. 4, 2811--2820; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64--68].

Cobalt↗
PubMed1979

Nuclear magnetic resonance studies on the tertiary folding of transfer ribonucleic acid: assignment of the 7-methylguanosine resonance.

Analysis of the low-field nuclear magnetic resonance (NMR) spectra of several class 1 D4V5 transfer ribonucleic acid (tRNA) species containing 7-methylguanosine in their variable loops reveals a set of six to seven tertiary base pair resonances, one of which is always located at ca. --13.4 ppm. Other tRNA species which do not contain 7-methyl-guanosine do not contain the tertiary resonance at --13.4 ppm. Chemical removal of 7-methylguanosine from several tRNAs containing the same dihydrouridine (DHU) helix sequence as yeast tRNAPhe results in the loss of the --13.4-ppm tertiary resonance. In the initiator methionine tRNA, which contains a different DHU helix sequence, the 7-methylguanosine hydrogen bond has been assigned at --14.55 ppm by chemical removal of this residue. In these experiments the aromatic C8H proton of 7-methylguanosine was also assigned (--9.1 ppm). The unexpectedly low-field position of the 7-methylguanosine resonance is explained by the deshielding effect of the delocalized positive charge in this nucleoside.

Base Composition↗
PubMed1979

Normal tRNAs promote ribosomal frameshifting.

The addition of Ser AGC AGU tRNA to an E. coli cell-free protein synthesizing system which contains the endogenous tRNA levels results in up to 100% of the ribosomes translating the MS2 coat gene shifting into the -1 reading frame. An analogous phenomenon is seen at a much lower level without the tRNA addition, where a shift into the +1 frame can also be detected. Thus translation with the endogenous tRNA levels yields proteins which have the amino terminus of the coat protein but which are substantially larger than the coat protein and comprise about 5% of the coat translation. Since the lysis gene overlaps the 3' end of the coat gene in the +1 frame, we conclude that the reading frame shift into the +1 frame yields a hybrid protein. Also, we present evidence that ribosomes translating the synthetase gene shift into the -1 frame near the distal end of the gene. This frameshifting is promoted by thrACU ACC tRNA. Specific competitor tRNAs for both Thr and Ser tRNA-promoted frameshifting have been characterized. The generality of this new mechanism for producing additional proteins is unclear, but it investigation should increase understanding of the coding mechanism and its origin.

Amino Acid Sequence↗
PubMed1977

A nuclear magnetic resonance study of secondary and tertiary structure in yeast tRNAPhe.

We present experimental evidence which confirms recently proposed ring current prediction methods for assigning hydrogen-bond proton nuclear magnetic resonance (NMR) spectra from tRNA (Robillard, G. T., Tarr, C. E., Vosman, F., & Berendsen, H. J. C. (1976) Nature (London) 262, 363-369; Robillard, G. T., Tarr, C. E., Vosman, F., & Sussman, J. L. (1977) Biophys. Chem. 6, 291-298). The evidence is a series of temperature-dependent studies on yeast tRNAPhe monitoring both the high- and low-field NMR spectral regions, which are correlated with independent optical and temperature-jump (temp-jump) studies performed under identical ionic strength conditions. Using assignments derived from the new prediction methods, the melting patterns of the hydrogen-bonded resonances agree with those expected on the basis of optical, temp-jump, and NMR studies on the high-field spectral region. The implication of these results is that previous assignment procedures are at least partially incorrect and, therefore, studies based on those procedures must be reexamined.

Anticodon↗
PubMed1977

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 1. Species containing a small variable loop.

Eight class I tRNA species have been purified to homogeneity and their proton nuclear magnetic resonance (NMR) spectra in the low-field region (-11 to -15 ppm) have been studied at 360 MHz. The low-field spectra contain only one low-field resonance from each base pair (the ring NH hydrogen bond) and hence directly monitor the number of long-lived secondary and tertiary base pairs in solution. The tRNA species were chosen on the basis of their sequence homology with yeast phenylalanine tRNA in the regions which form tertiary base pairs in the crystal structure of this tRNA. All of the spectra show 26 or 27 low-field resonances approximately 7 of which are derived from tertiary base pairs. These results are contrary to previous claims that the NMR spectra indicate the presence of resonances from secondary base pairs only, as well as more recent claims of only 1-3 tertiary resonances, but are in good agreement with the number of tertiary base pairs expected in solution based on the crystal structure. The tertiary base pair resonances are stable up to at least 46 degrees C. Removal of magnesium ions causes structural changes in the tRNA but does not result in the loss of any secondary or tertiary base pairs.

Binding Sites↗
PubMed1977

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 2. Species containing a large variable loop.

The number of base pairs in the solution structure of several class III D3VN tRNA species from E. coli has been determined by analyzing the number of low-field (-15 to -11 ppm) proton resonances in their nuclear magnetic resonance spectra at 360 MHz. Contrary to previous reports indicating the absence of tertiary resonances, all the spectra exhibit the expected number of secondary base pair resonances plus approximately ten extra resonances derived from tertiary base pairs in the three-dimensional folding of these molecules. The possible origins of some of these tertiary resonances are discussed; none of the spectra exhibits the characteristic resonance of the 8-14 tertiary base pair seen in class I D4V5 tRNA spectra.

Binding Sites↗
PubMed1977

NMR spectroscopy of the ring nitrogen protons of uracil and substituted uracils; relevance to A psi base pairing in the solution structure of transfer RNA.

The chemical shifts of the well-resolved ring nitrogen protons of uracil and a series of substituted uracils, including pseudouridine (5-ribosyl-uracil) and uridine (1-ribosyl-uracil) were determined using nuclear magnetic resonance (NMR). The observed chemical shifts suggest the existence of an atypical syn conformation for pseudouridine in the Apsi base pair in regulatory tRNAs in solution.

Hydrogen Bonding↗
PubMed1976

A study of secondary and tertiary solution structure of yeast tRNA(Asp) by nuclear magnetic resonance. Assignment of G.U ring NH and hydrogen-bonded base pair proton resonances.

The 270-MHz spectra of yeast tRNA(Asp) in H2O solutions containing Mg2+ show clearly resolved resonances in the region from -15 to -9.5 ppm. Resonances between -15 and -11.5 ppm from the hydrogen-bonded protons of the acceptor stem and anticodon arm decrease in intensity with increasing temperature and disappear by 75 degrees C. Simultansously, four well-resolved resonances between -11.2 and -10.3 ppm also decrease in intensity and disappear. Because of this behavior and their positions these resonances have been assigned to the four ring NH protons of G.U base pairs 5 and 30 in the acceptor stem and anticodon arm which are thereby shown not to be hydrogen bonded by normal Watson-Crick hydrogen bonds. The five G.C base pair resonances of the T psi C arm remain visible above 70 degrees C after all other resonances have disappeared. The high-temperature tRNA spectrum agrees well with that of the isolated T psi C hairpin and CCA half-molecule fragments, each of which contains the same five hydrogen-bonded proton resonances. The root-mean-square error between the observed and calculated resonance positions for the hydrogen-bonded base pair protons of these three arms is 0.19 ppm. The dihydrouridine stem is expected to have two A.U Watson-Crick base pairs and no B.C base pairs. However, it does not contribute any hydrogen-bonded resonances to the nuclear magnetic resonance (NMR) spectrum below -11.5 ppm. This suggests that even at 35 degrees C this helix is not hydrogen bonded in a normal manner. In the region below -11.4 ppm there are three additional proton resonances melting earlier than the rest which cannot be assigned to a particular helix of the cloverleaf. We suggest that these resonances arise from hydrogen-bonded protons involved in stabilizing tertiary structure.

Binding Sites↗
PubMed1975

Separation of transfer ribonucleic acid by sepharose chromatography using reverse salt gradients.

The transfer ribonucleic acids of Escherichia coli bind to unsubstituted Sepharose in the presence of high concentrations of ammonium sulfate at pH 4.5. Transfer RNA species are eluted individually from the Sepharose by a gradient from high to low concentrations of ammonium sulfate; leucine tRNA is fractionated into five isoaccepting species. The order of elution of these isoaccepting species differs from that seen with reverse phase chromatography. By means of only these two procedures, one isoaccepting species of leucine tRNA can be purified to apparent homogeneity. Isoaccepting tRNA species for 9 amino acids have been resolved. This established the general utility of this chromatographic system for the separation and purification of specific isoaccepting transfer RNAs.

Chromatography, Gel↗
PubMed1975

Tertiary hydrogen bonds in the solution structure of transfer RNA.

The high resolution nuclear magnetic resonance (NMR) spectra of hydrogen-bonded protons in four tRNAs have been studied at 270 MHz. The relative intensity of the resonances between -11 ppm and -15 ppm of Escherichia coli tRNA1-Va1 indicate that there are 26 plus or minus 3 protons, while only 20 are expected from secondary structure Watson-Crick hydrogen bonds inthe cloverleaf structure. Several possible candidates for these extra resonances are suggested by tertiary interactions observed in recent crystallographic studies. Of the four tRNAs studied, three, e.g., E. coli tRNA1Va1, E. coli tRNA-Arg and E. coli tRNA-Phe have one "GU pair" in their cloverleaf structure, while the fourth, yeast tRNA-Asp,has three "GU pairs" and one "G pair". Correlating these with the NMR spectra in the -10 ppm to -11 ppm region allows us to conclude that the "GU pairs" are not hydrogen-bonded by tautomerization to the lactim form. At the very low field region, near -14.9 ppm, the three E. coli tRNAs show a single resonance which is attributed to the 4-thiouracil 8 to adenine 14 hydrogen bond of the tertiary structure, by analogy with the recent crystal structure of yeast tRNA-Phe. This assignment is confirmed by the disappearance of this resonance after treatment with cyanogen bromide.

Arginine↗