Ribonucleic Acid Species Research Articles

In Vivo Aminoacylation of Transfer Ribonucleic Acid in Bacillus subtilis and Evidence for Differential Utilization of Lysine-Isoaccepting Transfer Ribonucleic Acid Species

The presence or absence of certain amino acids has different effects on the ability of Bacillus subtilis to sporulate, and the intracellular pool size of amino acids has been reported to vary during sporulation. The idea that these variations might exert a regulatory effect through aminoacylation of transfer ribonucleic acid (tRNA) was investigated by studying the levels of aminoacylation in vivo in the logarithmic or stationary phase of growth. Both the periodate oxidation method and the amino acid analyzer were used to evaluate in vivo aminoacylation. The results indicated that in general the level of aminoacylation of tRNA's remained constant through stage III of sporulation, although there were detectable variations for specific amino acid groups. Our studies also showed that periodate oxidation damaged certain tRNA's; therefore, the results obtained by such a method should be interpreted with caution. Because the damage can affect certain isoaccepting species specifically, the periodate oxidation method cannot be used to establish which isoaccepting species are acylated in vivo. We also investigated the possibility of preferential use of particular tRNA species by polyribosomes. These results demonstrated a preferential use of lysyl-tRNA's at different growth stages. Control mechanisms operating during the early stages of sporulation, therefore, do not affect the overall level of aminoacylation. However, there is an effect on the levels of aminoacylation of specific amino acids and on which isoaccepting species are utilized by the polyribosome system.

Resolution of ribonucleic acids by Sepharose 4B column chromatography.

Ribonucleic acids were resolved by molecular sieve chromatography on columns of Sepharose 4B. The elution positions of messenger ribonucleic acids were determined by detection of polyadenosine tracts and by support of protein synthesis in a messenger-dependent cell-free system. The elution position of other ribonucleic acid species from the Sepharose 4B was determined by formamide-sucrose density gradient centrifugation. Resolution of ribonucleic acids by this column was not dependent on molecular weight but rather on other properties such as secondary structure or the presence of poly(adenylic acid). The elution profiles of ribonucleic acids on cross-linked Sepharose 4B differed markely from those on conventional Sepharose and appeared to depend on molecular size alone. There was diminished resolution of high molecular weight ribonucleic acids on such columns.

Biochemical and regulatory properties of Escherichia coli K-12 hisT mutants

Escherichia coli K-12 hisT mutants were isolated, and their properties were studied. These mutants are derepressed for the histidine operon, map close to the purF locus at about 49.5 min on the E. coli linkage map, and lack pseudouridylate synthetase activity. The defect in this enzyme leads to the absence of pseudouridines in the anticodon loop of several transfer ribonucleic acid species, as evidenced by the altered elution profile on reversed-phase chromatography and resistance to amino acid analogues. Finally, the hisT mutants studied have a reduced growth rate that appears to be linked to hisT, although it is not known whether it is due to the same mutation. The normal generation time can be restored by supplementing the medium with adenine, uracil, and isoleucine.

Separation of isoacceptor cysteine transfer ribonucleic acids of bakers' yeasts

Isoacceptor species of certain amino acid-specific transfer ribonucleic acids (tRNAs) were fractionated by gel permeation chromatography using Sephadex G-100. The separation is attributed to the 20% ethanol-1% NaCl solvent and to the characteristics of Sephadex. Isoacceptor tRNAs specific for cysteine, arginine, phenylalanine, and histidine were recovered from commercial tRNA of yeast by this method. Highly purified cysteine-specific tRNA, obtained by a method which would not be expected to separate isoacceptor molecules when fractionated by this procedure, was shown to contain two cysteine isoacceptor tRNAs.

NOVEL Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiaion of a round of deoxyribonucleic acid replication

The initiation process of deoxyribonucleic acid (DNA) replication in Escherichia coli has been studied using the thermoreversible dna initiation mutant E. coli HfrHl65/120/6 dna-252. This dna mutation was incorrectly classed as a dnaA mutation. Biochemical and genetic evidence suggests that the dna-252 mutant is a novel dnaB mutant, possessing phenotypic properties which distinguish it from other dnaB mutants. Sensitivity of reinitiation in the dna-252 mutant to specific inhibitors of protein, ribonucleic acid (RNA), and DNA synthesis was studied. Reinitiation is shown to be sensitive to rifampin and streptolydigin but not to cholramphenicol. Thus, the dna-252 gene product appears to be required during the initiation process for a step occurring either before or during synthesis of an RNA species (origin-RNA). Using reversible inhibition of RNA synthesis by streptolydigin of a streptolydigin-sensitive derivative of the dna-252 mutant, the dna-252 gene product is shown to be directly involved in the synthesis of an orgin-RNA species. These results are included in a schematic model presented in the accompanying paper of the temporal sequence of events occurring during the initiation process.

Poly(uridylic acid) sequences in messenger ribonucleic acid of HeLa cells

The poly(uridylic acid [poly(U)] sequences of 30 to 40 nucleotides found in the heterogeneous nuclear ribonucleic acid (RNA) Of HeLa cells are also present in the poly(adenylic acid) [poly(A)] containing messenger RNA (mRNA) of these cells. Twenty-five percent of the total poly(U) sequences in the cell are found in the cytoplasm after HeLa cells have been labeled for 2.5 h with 32P. This value is close to 40% if only poly(A) containing RNA molecules of the cell are considered. The distribution of poly(U) sequences parallels that of poly(A) sequences in different size classes of cytoplasmic messenger RNA. From the concentrations and lengths of the two sequences it can be estimated that about 20% of the poly(A) containing mRNA molecules could contain one poly(U) sequence. Several lines of evidence have been developed to rule out poly(U) containing heterogeneous nuclear RNA (HnRNA) as the source of the poly(U) sequences in cytoplasm. Poly(U) sequences are also found in RNA molecules of the nucleus and cytoplasm which do not contain poly(A). Although poly(U) sequences are most abundant in nuclear RNA species lacking poly(A), they are well represented in a class of RNA molecules in cytoplasm which lack poly(A), but which otherwise resemble the polysomal mRNA of HeLa cells.

Messenger ribonucleic acid metabolism in mammalian mitochondria. Discrete poly(adenylic acid) lacking messenger ribonucleic acid species associated with mitochondrial polysomes.

The mRNA species released from mitochondrial polysomes prepared by the Mg2+ precipitation technique have been further characterized using various analytical techniques. Mitochondrial polysomes were dissociated by treatment with puromycin and chemically labeled with (3H) dimethyl sulfate. About 51% of steady-state mitochondrial mRNA bind to oligo(dT)-cellulose indicating the presence of poly(adenylic acid)(poly(A)) in this fraction. The poly(A)-containing mRNAs resolve into discrete bands of 9-16 Se, while the RNA fraction unable to bind to oligo(dT)-cellulose representing poly(A)-lacking mRNA contains 8-12 Se species. About 90% of poly(A) lacking RNA hybridizes with mitochondrial DNA and less than 7% hybridizes with nuclear DNA. The extent of hybridization of poly(A)-lacking RNA with mitochondrial DNA was not significantly affected by the presence of excess mitochondrial rRNA, cytoplasmic rRNA, or a tenfold concentration of poly(A)-containing RNA isolated from total mitochondrial RNA. Possible differences in sequence properties between poly(A)-containing and -lacking mitochondrial mRNAs were further verified using a solid phase-bound cDNA procedure. Poly(A)-containing mRNA released from mitochondrial polysomes shows over 85% sequance homology with oligo(dT)-cellulose-bound cDNA prepared against total mitochondrial poly(A)-lacking mitochondrial mRNA hybridizes with the cDNA providing direct evidence for the distinct sequence properties of the two mRNA species.

Study of tyrosine transfer ribonucleic acid modification in relation to sporulation in Bacillus subtilis

A reversal in the relative amounts of the two major species of tyrosine transfer ribonucleic acid (tRNATyr) (I and II) has been previously observed by others during the development of Bacillus subtilis. These species have been purified by benzoylated diethylaminoethyl-cellulose chromatography and were shown to differ by the modification of an adenosine residue (species I contains i6A and species II ms2i6A). As suggested by competitive hybridization assays, they might possess the same nucleotide sequence. A tRNATyr species lacking isopentenyl and methylthio moieties was not detected. The structural difference between species I and II was shown to be important for ribosome binding but not for charging. The extent of alteration during growth was studied in parallel with physiological events. Like sporulation, tRNATyr change is iron dependent. Moreover, when sporulation is prevented by an excess of glucose, the tRNATyr change is delayed as is the synthesis of enzymatic systems required for the onset of sporulation. tRNATyr change also demands unceasing protein synthesis.

Ribonucleic acid synthesis in normal and immune macrophages after antigenic stimulus

Macrophage ribonucleic acid (RNA) synthesis is an important metabolic process intimately related to the function of these cells. Mouse peritoneal macrophage RNA was extracted with phenol in the presence of bentonite and electrophoresed on composite agarose-polyacrylamide gels. The pulse-chase technique was used to follow the precursor relationships in macrophage ribosomal RNA (rRNA) maturation. The rRNA species at 18S and 28S appeared at 15 and 45 min, respectively, after RNA synthesis was halted. Their appearance corresponded closely to decreases in the rRNA precursors at 45S, 36S, and 34S. Studies of RNA methylation aided in confirming the identity of these ribosomal species. Unmethylated RNA species appeared as messenger RNA between 5S and 15S, and at about 55S probably represented heterodisperse nuclear RNA. When normal macrophages were incubated with heat-killed Salmonella enteritidis, an acceleration in the maturation of RNA was observed. The accelerated maturation was indicated by the earlier appearance of 28S rRNA and the more rapid development of an equilibrium state, where further labeling did not change the RNA profile. In macrophage RNA from mice immunized with S. enteritidis, rRNA species appeared rapidly but did not accumulate to the same extent as observed for normal macrophages. Precursor rRNA and other RNA species developed as usual, suggesting specific degradation of mature rRNA. Such rRNA wastage could indicate a mechanism controlling ribosome assembly in the non-proliferating activated macrophage. The pattern of RNA synthesis in immune macrophages was essentially unchanged by the presence of heat-killed S. enteritidis in vitro.

Nonribosomal nature of novel, stable ribonucleic acid species accumulated by blue-green bacteria.

The facultatively chemoheterotrophic blue-green bacterium Aphanocapsa 6714 accumulates two novel, stable ribonucleic acid species when deprived of sources of carbon and energy. At least one of these species is nonribosomal.

Isolation and characterization of messenger ribonucleic acid species for guinea-pig milk proteins from free and membrane-bound polyribosomes.

Morphological and cytochemical evidence has suggested the existence of two distinct classes of polyribosomes in secretory cells, those attached to the endoplasmic reticulum (bound polyribosomes) and those apparently free in the cytoplasmic matrix (free polyribosomes). Siekevitz & Palade (1960) proposed that in secretory cells, membranebound polyribosomes were engaged primarily in the synthesis of proteins for export, whereas free polyribosomes synthesized primarily proteins for intracellular requirements. To study the functional specificity of the two polyribosomal classes it is necessary to isolate undegraded polyribosomes and to demonstrate in vitro the ability or otherwise of their mRNA species to direct the synthesis of specific secretory proteins. Free and membrane-bound polyribosomes were isolated from lactating guinea-pig mammary gland by differential centrifugation (see Fig. l ) , essentially as described by Blobel & Potter (1967). The criteria for the successful preparation of intact polyribosomes were based on our ability to isolate undegraded mRNA from the preparations, as judged both by polyacrylamide-gel electrophoresis under denaturing conditions and the ability of these mRNA species to direct the synthesis of protein in cell-free translational systems. The separation of the polyribosomal classes involved a prolonged centrifugation, increasing the susceptibility, particularly of the membrane-bound polyribosomes, to degradation by intracellular ribonuclease activities. In order to inhibit such activities all isolation solutions contained heparin (250,ug/ml) and 0.2~01. of guinea-pig liver high-speed supernatant. Examination of polyribosomes isolated in this manner on 1040% sucrose gradients revealed high-molecular-weight polyribosomal species with little evidence of monoribosomes, diand tri-mers. Similar profiles were obtained from both free and membranebound polyribosomal preparations mRNA isolated from both polyribosomal fractions by chromatography on oligo(dT)-cellulose directed the synthesis of protein in a cell-free system derived from Krebs I1 ascites cells. Immunoprecipitation with antibodies specific for the guinea-pig-milk casein fraction (Table 1 ) clearly demonstrated the presence of mRNA species for secretory proteins in both free and bound polyribosomal fractions. However, on the basis of units recovered after phenol/chloroform extraction, 15 %

Thermosensitive mutations affecting ribonucleic acid polymerases in Saccharomyces cerevisiae.

Among 150 temperature-sensitive Saccharomyces cerevisiae mutants which we have isolated, 15 are specifically affected in ribonucleic acid (RNA) synthesis. Four of these mutants exhibit particularly drastic changes and were chosen for a more detailed study. In these four mutants, RNA synthesis is immediately blocked after a shift at the nonpermissive temperature (37 C), protein synthesis decays at a rate compatible with messenger RNA half-life, and deoxyribonucleic acid synthesis increases by about 40%. All the mutations display a recessive phenotype. The segregation of the four allelic pairs ts-/ts+ in diploids is mendelian, and the four mutants belong to three complementation groups. The elution patterns (diethylaminoethyl-Sephadex) of the three RNA polymerases of the mutants grown at 37 C for 3.5 h show very low residual activities. The in vitro thermodenaturation confirms the in vivo results; the half-lives of the mutant activities at 45 C are 10 times smaller than those of the wild-type enzymes. Polyacrylamide gel electrophoresis shows that the synthesis of all species of RNA is thermosensitive. The existence of three distinct genes, which are each indispensable for the activity of the three RNA polymerases in vivo as well as in vitro, strongly favors the hypothesis of three common subunits in the three RNA polymerases.

Characterization of 5.8S ribosomal ribonucleic acid in Neurospora crassa.

Neurospora crassa ribosomes contain a species of ribonucleic acid (RNA) of molecular weight 54,000, similar to 5.8S ribosomal RNA previously described for other eukaryotic organisms. The 5.8S RNA from N. crassa was found to be released by heat treatment at 60 C from 25S ribosomal RNA but not from 18S ribosomal RNA. The base composition of N. crassa 5.8S RNA was similar to that of 5.8S RNA from Saccharomyces cerevisiae, but differed from animal 5.8S RNA. During the course of this study, it was discovered that N. crassa 25S ribosomal RNA had a number of internal cleavages that may exist in vivo.

Effect of growth rate on the amounts of ribosomal and transfer ribonucleic acids in yeast.

The steady-state growth rate of Saccharomyces cerevisiae was varied by growing the cells in different media. The total amount of ribonucleic acid (RNA) per cell was found to decrease as a nonlinear function of decreasing growh rate. The RNA from cells growing in different media was analyzed by polyacrylamide gel electrophoresis. Although the amounts of both ribosomal RNA and transfer RNA decreased with decreasing growth rate, the ratio of ribosomal to transfer RNA was not constant. As the growth rate was reduced the ribosomal RNA fraction decreased slightly, whereas the transfer RNA fraction increased slightly. Thus the levels of ribosomal and transfer RNA were regulated to similar yet different extents. The levels of the different ribosomal RNA species were more closely coordinated. At all growth rates the ribosomal RNAs (including 5S RNA) were present in equimolar amounts. The rate of protein synthesis in yeast cells also decreased with decreasing growth rate. The low rates of protein synthesis did not appear to be due to limiting numbers of ribosomes or transfer RNA molecules.

Multiple gene loci for a single species of glycine transfer ribonucleic acid.

The study of suppressors of tryptophan synthase A protein missense mutations in Escherichia coli has led to the establishment of two nonadjacent genetic loci (gly V and gly W) specifying identical nucleotide sequences for a single isoaccepting species of glycine transfer ribonucleic acid (tRNA GLY 3 GGU/C). In one case, suppression of the missense mutation trpA78 was due to a mutation in a structural gene (gly W) for tRNA Gly 3 GGU/C. This mutation resulted in a base change in the anticodon and modification of an A residue adjacent to the 3' side of the anticodon, leading to the production of a tRNA Gly 3 UGU/C species. The resulting glyW51 (SU UGU/C) allele was mapped by interrupted mating and was located at approximately 37 min on the Escherichia coli genetic map. Other suppressor mutations affecting the primary sequence of tRNA Gly GGU/C and giving rise to the Ins and SU+A58 phenotypes were positioned at 86 min (glyV). Several independently arising missense suppressor mutations resulting in the SU+A78 phenotypes were isolated and mapped at these two genetic loci (glyV and glyW). The ratio of appearance of suppressor mutations at glyV and glyW suggests that there are three of four tRNAGly3 GGU/C structural gene copies at the glyV locus to one copy at the glyW locus. Structural genes for tRNA ly isoacceptors are now known at four distinct locations on the Escherichia coli chromosome: glyT (77 MIN), TRNA Gly 2 GGA/G; gly U (55 min), tRNAGly-1 minus; and gly V (86 MIN) AND GLYW (37 min), tRNAGly 3 GGU/C.

Alteration of tyrosine isoaccepting transfer ribonucleic acid species in wild-type and asporogenous strains of Bacillus subtilis.

The relative amounts of two isoacceping species of tyrosine transfer ribonucleic acid, tRNATyrI and tRNATyrII, determined from reversed phase 5 profiles of tyrosyl-tRNA, prepared from Bacillus subtilis strain W168, were growth phase and medium dependent. The growth phase-dependent alterations in the relative amounts of tRNATyr species were also demonstrated in 11 asporogenous strains of B. subtilis. The proportion of tRNA-Tyr species and the extent of the alteration in their relative amounts during the transition from the exponential to the stationary phase of growth of these strains was not directly correlated with the formation of spores by strain W168 grown in various media or the stage at which the asporogenous strains are blocked in the process of sporulation.

Stability of ribosomal and transfer ribonucleic acid in Escherichia coli B/r after treatment with ethylenedinitrilotetraacetic acid and rifampicin.

A short treatment with ethylenedinitrilotetraacetic acid to permeabilize bacteria for various antibiotics or treatment with the ribonucleic acid (RNA) synthesis inhibitor rifampin causes a slow degradation of 50S and 30S ribosomal particles and of the corresponding 23S and 16S ribosomal RNA species (about 25 percent in 1 h). The effects are additive such that the decay is about 50 percent/h if rifampin is employed after permeabilization by ethylenedinitrilotetraacetic acid. The 5S ribosomal RNA and transfer RNA are essentially stable under these conditions.

New transfer ribonucleic acid species during sporulation of Bacillus subtilis.

The transfer ribonucleic acid (tRNA) populations from log-phase cells, sporulating cells (stage III), and dormant spores were compared by tRNA-deoxyribonucleic acid hybridization techniques. New tRNA species not found in log-phase cells were observed in stage III cells. Some of the tRNA made during sporulation were also present in dormant spores. Although the role and function of these new tRNA species cannot be ascribed directly to the sporulation process, their presence indicates that new tRNA genes can be transcribed during sporulation and suggests that translational control may be exerted during sporulation by tRNA.

Ribonucleic acid synthesis in streptomyces antibioticus: Stable ribonucleic acid species synthesized by young and old cells

In studies of RNA synthesis by intact cells and cell-free extracts of Streptomyces antibioticus , it has been found that 48 hr cells (producing actinomycin) and cell-free extracts are less efficient than 12 hr cells (not producing actinomycin) and extracts in the synthesis of RNA. Analysis of the products of “in vivo” and “in vitro” RNA synthesis by sucrose gradient centrifugation reveals that both 12 and 48 hr cultures and cell-free extracts synthesize ribosomal RNA as well as RNA species of higher and lower molecular weights. However, 50–60% of the 3H-uridine labelled RNA synthesized by intact cells sediments as rRNA as compared with only 5–10% of the cell-free product. The addition of 2 × 10 −5 M actinomycin D to incubation mixtures for cell-free RNA synthesis does not significantly alter the relative amounts of the various RNA species synthesized by 12 or 48 hr extracts.

Transfer of serine into polypeptides and myosin by chromatographic species of seryl-transfer ribonucleic acid.

The efficiencies of two chromatographic species of [3-H]seryl-tRNA, namely peaks I and II, in cell-free amino acid incorporation were investigated. The maximum yield of polypeptide seems to be the same for the reaction mixtures containing either peak I or peak II, suggesting that the efficiency of both peaks in total protein synthesis is the same. The efficiency of transfer of serine into myosin heavy subunit (myosin H) by peaks I and II was also investigated. Peak II of [3-H]seryl-tRNA transfers three times as much serine into myosin H as peak I.