Determination of base pairing in yeast 5S and 5.8S RNA infrared spectroscopy.

Infrared Spectroscopy was used to determine the numbers of base pairs for yeast 5S RNA and 5.8S RNA. The spectra were recorded at 20 degrees C and 50 degrees C, where tertiary interactions are assumed to be of less importance. It may be concluded that the structure of both RNAs is highly ordered and that there are large contributions of tertiary interactions. The results are compared with data derived from structural models that were proposed in the literature as well as with data previously published for prokaryotic 5S RNAs.     

Binding oligonucleotides to Escherichia coli and Bacillus stearothermophilus 5 S RNA.

Binding complementary tri- and tetranucleotides to Escherichia coli A19 and Bacillus stearothermophilus 799 5 S RNAs permitted identification of single-stranded regions in these RNAs. Sequences around positions 10, 30, 60, 70, 85 and 95 are in a single-stranded conformation in both 5 S RNAs. It is concluded that the overall structure of bacterial 5 S RNA has been conserved during evolution. Two types of structural conservation have been observed at specific sites of the 5 S RNA: firstly, nucleotide sequence and single strandedness and secondly, single strandedness only. The oligonucleotide binding data for E. coli 5 S RNA are in general agreement with a previous study (Lewis and Doty, 1970) and do not support fully any proposed structural model.     

Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.

Summary E. coli [³²P]-labelled 5S RNA was complexed with E. coli and B. stearothermophilus 50S ribosomal proteins. Limited T₁ RNase digestion of each complex yielded three major fragments which were analysed for their sequences and rebinding of proteins. The primary binding sites for the E. coli binding proteins were determined to be sequences 18 to 57 for E-L5, 58 to 100 for E-L18 and 101 to 116 for E-L25. Rebinding experiments of purified E. coli proteins to the 5S RNA fragments led to the conclusion that E-L5 and E-L25 have secondary binding sites in the section 58 to 100, the primary binding site for E-L18. Since B. stearothermophilus proteins B-L5 and BL22 were found to interact with sequences 18 to 57 and 58 to 100 it was established that the thermophile proteins recognize and interact with RNA sequences similar to those of E. coli. Comparison of the E. coli 5S RNA sequence with those of other prokaryotic 5S RNAs reveals that the ribosomal proteins interact with the most conserved sections of the RNA.Paper number 12 on structure and function of 5S RNA.Preceding paper: Wrede, P. and Erdmann, V.A. Proc. Natl. Acad. Sci. USA 74, 2706–2709 (1977)     

Determination of the base pairing content of ribonucleic acids by infrared spectroscopy

A method is described for determining the fractions of adenine-uracil and guanine-cytosine base pairs of partly double-helical ribonucleic acids in aqueous solution. The method is based upon the ability to distinguish between paired and unpaired bases by means of infrared spectroscopy of their deuterium oxide solutions in the 1500–1800 cm^?1 frequency region of the infrared spectrum. An application of the method to yeast ribosomal RNA is described. At 30°C, ribosomal RNA is 64± 6% paired (35% GC, 29% AU). The method can be applied to determine the fractions of Watson-Crick base pairs at a given temperature in any RNA containing the four common bases in known ratios.     

Optical properties and base pairing of E. coli 5S RNA

The ultraviolet absorption, optical rotatory dispersion, circular dichroism, and infrared absorption spectra of renatured 5S RNA have been measured at pH 7.0 in 0.1M NaCl at 25° and used to obtain four independent estimates of the number of base pairs. These four estimations are in reaonable agreement and average values of 28 ± 4 G.C and 13 ± 4 A.U. base pairs.     

Correlation between 30S ribosomal proteins of Bacillus stearothermophilus and Escherichia coli

Summary The 30S ribosomal proteins from Bacillus stearothermophilus strains 799 and 10 were purified and correlated with those from E. coli by comparing their two-dimensional electrophoretic mobility, immunological cross-reaction, molecular weight, amino acid composition and partial amino acid sequence. A high degree of similarity was observed among the proteins from these taxonomically distant bacterial species.Paper No. 82 on Ribosomal proteins-preceding paper is by J. Horne and V. A. Erdmann, FEBS Letters, in press.N.R.C.C. No. 13514.     

Determination of base pairing in ribonucleic acid by Fourier-transform infrared spectrometry: yeast ribosomal 5S ribonucleic acid

Fourier-transform infrared (FT-IR) spectra of yeast ribosomal 5S RNA have been acquired at several temperatures between 30 and 90 degrees C. The difference spectrum between 90 (bases unstacked) and 30 degrees C (bases stacked) provides a measure of base stacking in the RNA. Calibration difference spectra corresponding to stacking of G-C or A-U pairs are obtained from "reference" FT-IR spectra of poly(rG) X poly(rC) minus 5'-GMP and 5'-CMP or poly(rA) X poly(rU) minus 5'-AMP and 5'-UMP. The best fit linear combination of the calibration G-C and A-U difference spectra to the 5S RNA (90-30 degrees C) difference spectrum leads to a total of 25 +/- 3 base pairs (17 G-C pairs + 8 A-U pairs) for the native yeast 5S RNA in the absence of Mg2+. In the presence of Mg2+, an additional six base pairs are detected by FT-IR (one G-C and five A-U). FT-IR melting curve midpoints show that A-U and G-C pairs melt together (65 and 63 degrees C) in the presence of Mg2+ but A-U pairs melt before G-C pairs (47 vs. 54 degrees C) in the absence of Mg2+.     

Overproduction and single-step purification of Bacillus stearothermophilus peroxidase in Escherichia coli

Summary The cloned peroxidase gene from Bacillus stearothermophilus was highly expressed in Escherichia coli. Using the high copy number plasmid which is temperature-sensitive and its own strong promoter, this thermostable peroxidase was produced at 28% of the total cell proteins when the cells were grown at 42°C. The enzyme could be easily purified from E. coli by heat treatment and single-column Sephadex G-200 chromatography. From a 200 ml culture, 30 mg of purified enzyme was obtained. The peroxidase produced by E. coli showed a thermostability, haem type and content identical with those of the peroxidase produced by B. stearothermophilus.Offprint requests to: H. Okada     

Base pairing in Bacillus subtilis ribosomal 5S RNA as measured by ultraviolet absorption and Fourier-transform infrared spectrometry

Ultraviolet (260 and 280 nm) and Fourier-transform infrared (FT-IR) spectra of Bacillus subtilis ribosomal 5S RNA have been acquired between 20 and 90 degrees C. In the presence of added Mg2+, the average UV melting midpoint, Tm, is 60 (A260) or 62 degrees C (A280), resolving into two components (Tm = 54 and 68 degrees C). In the presence of 10 mM Mg2+, the normalized A260 increases by about 5%, and the average Tm increases to 70 degrees C (A260 or A280), resolving into components at 63 and 73 degrees C at 260 nm but not resolved at 280 nm. From the difference of the 5S RNA FT-IR spectra between 90 and 30 degrees C, the number of base pairs in B. subtilis 5S RNA was determined by the procedure outlined in the accompanying paper [Li, S.-J., Burkey, K. O., Luoma, G. A., Alben, J. O., & Marshall, A. G. (1984) Biochemistry (preceding paper in this issue)]. Addition of 10 mM Mg2+ increases the number of A-U pairs by 1 (from 11 to 12) and the number of G-C pairs by 2 (from 15 to 17). FT-IR melting curve midpoints show that addition of Mg2+ increases the melting point for both A-U and G-C pairs in B. subtilis 5S RNA. The A-U pairs melt before G-C pairs (56 vs. 64 degrees C) in the absence of Mg2+, but both types of pairs melt at the same temperature (67 vs. 70 degrees C) in the presence of Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal denaturation and loss of viability in Escherichia coli and Bacillus stearothermophilus

When Escherichia coli was heated at 10°C/min in a differential scanning calorimeter, the onset of irreversible thermal denaturation occurred at 51°C, about 5°C above the maximum growth temperature. The temperature at which death rate was maximal (63°C) coincided with the thermogram peak caused by denaturation of the 30S ribosomal subunit. The maximum death rate in vegetative cells of Bacillus stearothermophilus occurred at the higher temperature of 71°C which also coincided with the leading edge of the main thermogram peak.     

Sequence homologies between the tryptophanyl tRNA synthetases of Bacillus stearothermophilus and Escherichia coli.

Subcultures of ovaries and testis of the crab Carcinus maenas have been performed in the presence of L-[Me-¹⁴C]methionine. Introduction in the medium of a chromatographically-purified liposoluble fraction from the androgenic glands of the same animal inhibits the biological methylation of the tRNA of the ovaries by 62%. The inhibition of methylation of five individual bases varies from 45% to 84%. No inhibition of tRNA methylation is observed under the same conditions with testis subcultures.     

Bacillus stearothermophilus plasmid pSTK1 replicon is functional in Escherichia coli

Summary A kanamycin-resistant plasmid possessing a thermostable replicon derived from Bacillus stearothermophilus cryptic plasmid pSTK1 was constructed. The plasmid could transform not only B. stearothermophilus and Bacillus subtilis, but also Gram-negative Escherichia coli. The behavior of the plasmid in the hosts was examined. The plasmid was stably maintained even at 67°C in B. stearothermophilus without selective pressure. During the plasmid replication, single-stranded DNA (ssDNA) intermediates were found in E. coli, while these were not found in B. subtilis.     

Amino-Terminal Sequences of Some Escherichia coli 30S Ribosomal Proteins and Functionally Corresponding Bacillus stearothermophilus Ribosomal Proteins

Amino-terminal sequences of five purified Escherichia coli 30S ribosomal proteins (S4, S9, S10, S16, and S20) were compared with those of their functionally corresponding Bacillus stearothermophilus ribosomal proteins identified previously by the reconstitution technique. An automatic Edman degradation method was used for sequence determinations. The sequence of the first 30 residues is presented, except that only the first 25 residues are shown for the S20 pair. Substantial (40 to 70%) sequence homologies have been observed in every case. The results show that the pairs of functionally equivalent proteins, previously identified by the reconstitution technique, are also chemically related. Thus, the present chemical studies give further support for the previous conclusion that two ribosomes with different properties, 30S subunits from E. coli and B. stearothermophilus, have the same fundamental structural organization.     

Base cleavage specificity of angiogenin with Saccharomyces cerevisiae and Escherichia coli 5S RNAs

The base cleavage specificity of angiogenin toward naturally occurring polyribonucleotides has been determined by using rapid RNA sequencing technology. With 5S RNAs from Saccharomyces cerevisiae and Escherichia coli, angiogenin cleaves phosphodiester bonds exclusively at cytidylic or uridylic residues, preferably when the pyrimidines are followed by adenine. However, not all of the existent pyrimidine bonds in the 5S RNAs are cleaved, likely owing to elements of structure in the substrate. Despite the high degree of sequence homology between angiogenin and ribonuclease A (RNase A), which includes all three catalytic as well as substrate binding residues, the cleavage patterns with natural RNAs are unique to each enzyme. Angiogenin significantly hydrolyzes certain bonds that are not appreciably attacked by RNase A and vice versa. The different cleavage specificities of angiogenin and RNase A may account for the fact that the former is angiogenic while the latter is not.