Brian R. Reid

Ring-current shifts in the 300 MHz nuclear magnetic resonance spectra of six purified transfer RNA molecules

Abstract We present the 300 MHz high-resolution proton nuclear magnetic resonance spectra of the ring NH hydrogen-bonded protons of six purified tRNAs. Good agreement was obtained between the observed spectra and those computed on the assumption of the suitable cloverleaf models. In the computation it is assumed that the hydrogen-bonded ring NH in each type of base pair has an intrinsic position with respect to 2,2-dimethyl-2-silapentane-5-sulfonate, i.e. in A·U it is at −14·8 parts per million, in G·C at −13·7 parts per million and in A·Ψat −13·5 parts per million. The shifts of these resonances from these positions are calculated by including ring current fields from the nearest neighbors. The agreement is very good, adding support to our earlier findings that there is no evidence for additional Watson-Crick base pairs detected beyond those in the cloverleaf. In general, resolved resonances are fitted by the computed spectra to within ±0·2 part per million showing that there is no need for any additional physical mechanism to explain the nuclear magnetic resonance positions. Hence, the nuclear magnetic resonance spectra can be interpreted in terms of the structure of their neighbors and in a few important cases this has been particularly valuable in understanding the structure beyond the end of a helical region. In the tRNA Glu E.coli′ for example, the positions of the resonances in A·U no. 7 and A·U no. 49 at the interior ends of the acceptor and -T-Ψ-C- stems, respectively, strongly suggest that these two stems are in a continuous helix. Other structural effects at the ends of the helical regions are also suggested by the nuclear magnetic resonance spectra.

Assignment of the low field proton nuclear magnetic resonance spectrum of yeast phenylalanine transfer RNA to specific base pairs

Abstract High-resolution proton nuclear magnetic resonance spectra at 220 and 300 MHz have been used to investigate the base-pairing structure of fragments of yeast tRNAPhe, of chemically modified tRNAPhe and of intact tRNAPhe. To a very good approximation the positions of the fragment spectra are additive within 0·2 part per million, indicating that factors responsible for certain structural features in the intact molecule are already present in the smaller fragments (half molecules, hairpins and 3 4 molecules). A simple first-order ring-current shift theory taken in conjunction with the cloverleaf model for tRNAPhe (RajBhandary et al., 1967) has been used to predict the low-field (− 15 to −11 part per million) nuclear magnetic resonance spectra and make assignments of the resolved resonances to ring NH protons of specific base pairs. The general agreement between the predicted and observed spectra to within 0·2 part per million confirms in detail the cloverleaf model for the secondary structure of tRNAPhe in solution. It is also established that ring-current shifts are the principal factor responsible for the wide range of shifts observed in the low-field spectra. As a result it is evident that the resonances are very sensitive to small changes in the secondary structure and in some cases changes in the interbase distance as small as 0·2 A could easily be detected. It is also clear from the analysis that certain of the resonances are sensitive to the tertiary structure of the molecule and specific examples are discussed. As with our previous study, we find no evidence for any strong Watson-Crick type base pairs beyond those predicted by the cloverleaf structure.

Investigation of exchangeable protons and the extent of base pairings in yeast phenylalanine transfer RNA by high resolution nuclear magnetic resonance

Abstract High resolution nuclear magnetic resonance spectra were obtained for yeast transfer RNA Phe and unfractionated yeast tRNA in aqueous solutions. Resonances due to the free amino protons, hydrogen-bonded amino protons and hydrogen-bonded ring N—H protons were observed in three non-overlapping spectral regions. The assignment of the exchangeable proton resonances in yeast tRNA Phe is based on their pH and temperature dependence, spectral position, and their disappearance in deuterium oxide solvent. The number of each kind of exchangeable proton was determined and compared with the number expected on the basis of the cloverleaf model. The total number of base pairs observed by nuclear magnetic resonance (20±2) agrees well with the number computed from the cloverleaf model (20±1) depending upon whether or not one 2MeG.C and one G.U base pair are included. Furthermore, the separate numbers of A.U and G.C base pairs, 7 to 8 and 11 to 12, respectively, determined by nuclear magnetic resonance, are in good agreement with the 8 and 12 respective pairs expected from the cloverleaf model. The nuclear magnetic resonance spectra provided little evidence for additional strong Watson-Crick type base pairs resulting from tertiary structure such as are proposed in several models of tRNA. Resonances observed in the −9 to −11 parts per million region appear to be related to tertiary structure, but their assignment remains open at this tune. The results obtained with unfractionated yeast tRNA parallel those obtained with yeast tRNA Phe and therefore indicate that the interpretation of these latter data may be applicable to other tRNA species. The high resolution nuclear magnetic resonance data on the behavior of the exchangeable protons in tRNA are compared with the results of recent tritium exchange experiments. The anomalous behavior of hydrogen-bonded amino protons in both the nuclear magnetic resonance and tritium exchange experiments is discussed.

Helix-coil dynamics in RNA: the amino acid acceptor helix of Escherichia coli phenylalanine transfer RNA.

Abstract The RNA helix is a fluctuating dynamic structure in solution. In this paper we report a kinetic description of the individual base-pairs in an RNA helix (the acceptor stem of Escherichia coli tRNA Phe ) obtained by proton nuclear magnetic resonance spectroscopy using the saturation recovery techniques described by Johnston & Redfield (1977). We have determined the helix opening rates of each of the six Watson-Crick G·C base-pairs in the acceptor stem of partially unfolded E. coli tRNA Phe at elevated temperature in the presence of buffers, under which conditions saturation recovery is exchange-dominated and the exchange process is rate-limited by helix-coil opening.

The extent of base pairing in 5 s RNA Yeast 5 s RNA

Abstract The total number of secondary structure base pairs in yeast 5 s RNA has been determined by measuring the integrated intensities of the proton nuclear magnetic resonance in the −11 to −15 part per million region. In the absence of Mg2+ 21 ± 2 base pairs were observed at 35 °C. After incubating in the presence of Mg2+ the total base-pair content in yeast 5 s RNA increased to 28 ±3, most of which (85%) are G.C pairs. The small fraction of base pairing indicated by the nuclear magnetic resonance experiments is supported by comparative enzymic digestion studies which show that yeast 5 s RNA is considerably more susceptible to degradation than is transfer RNA.

Loop accessibility in transfer RNA.

Summary Endonuclease digestion of tRNA and unfractionated tRNA mixtures results in only discrete fragment sizes which are quarters or multiples thereof, thus strongly supporting the validity of the cloverleaf structure for all tRNAs. Kinetic studies with individual tRNA species and with unfractionated tRNA using pancreatic RNase as well as RNase T1 indicate that the anticodon loop is by far the most accessible region of the tRNA structure, being at least ten times more susceptible to cleavage than either side loop. RNase T1 fragmentation kinetics of species containing free guanine in one, two or all three major loops reveal that the dihydrouridine loop is the second most susceptible to attack, being several times more accessible than the ribothymidine loop. This has been confirmed with unfractionated tRNA and appears to be a general feature of tRNA structure in solution.

Selective inactivation of E. coli tRNA by ethylenimine.

Abstract Studies in this laboratory on the S-aminoethylation of cysteine by ethylenimine while attached to transfer RNA (tRNA) led to the observation that the ability of E. coli B tRNA to accept certain amino acids was abolished by exposure to this reagent. The demonstration of limiting amounts of thiopyrimidines, predominantly 4-thiouridine, in E. coli tRNA by Lipsett (1965) led us to consider the S-aminoethylation of thionucleotides as a possible explanation for the selective inactivation of amino acid acceptance by ethylenimine. The present communication demonstrates that aminoethylation by ethylenimine of 4-thiouridine residues in E. coli tRNA does occur under conditions mild enough to avoid covalent rupture of tRNA molecules. The kinetics of alkylation of 4-thioU residues as well as the kinetics of inactivation of acceptance for four different amino acids are reported.

Study of tRNA fragment interactions by Sephadex chromatography

Abstract Studies on the interaction of large fragments of yeast tRNAPhe are reported. Aggregation of these fragments and restoration of amino acid acceptor activity can be conveniently studied on Sephadex G-100 columns at room temperature in the presence of Mg++. All data can be presented in a single composite graph, the “difference” or “interaction” profile which at once and directly reveals reconstitution of size and function.

Fractionation of yeast ribosomal proteins.

Abstract Isolation and characterization of individual ribosomal proteins of E. coli has been reported by Traut et al. (1967). This communication is to report on the fractionation and analysis of proteins isolated from the ribosomes of brewers yeast. It was found that, as in the case of E. coli, the protein moiety of the yeast ribosome is made up of several distinct molecular species. Individual ribosomal protein species have been isolated by preparative gel electrophoresis, characterized by analytical gel electrophoresis and in most cases by amino acid analysis.

Isolation of tRNAyeastPhe half-molecules by gel filtration and chromatography on benzoylated deae-cellulose at high temperature

Abstract A mixture of half-molecules of tRNA Phe from yeast, produced by chemical cleavage of the anticodon loop, was isolated by gel filtration on Sephadex G-100 at 57° and fractionated by column chromatography on benzoylated DEAE-cellulose at the same temperature. Rechromatography under identical conditions yielded two major and a minor form of the 3′-half-molecule and a major and a minor form of the 5′ half-molecule. No pronounced differences were found in the amino acid acceptor activities of all complementary fragment combinations.