6S RNA regulates E. coli RNA polymerase activity

The E. coli 6S RNA was discovered more than three decades ago, yet its function has remained elusive. Here, we demonstrate that 6S RNA associates with RNA polymerase in a highly specific and efficient manner. UV crosslinking experiments revealed that 6S RNA directly contacts the sigma70 and beta/beta' subunits of RNA polymerase. 6S RNA accumulates as cells reach the stationary phase of growth and mediates growth phase-specific changes in RNA polymerase. Stable association between sigma70 and core RNA polymerase in extracts is only observed in the presence of 6S RNA. We show 6S RNA represses expression from a sigma70-dependent promoter during stationary phase. Our results suggest that the interaction of 6S RNA with RNA polymerase modulates sigma70-holoenzyme activity.     

6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter

6S RNA is an abundant noncoding RNA in Escherichia coli that binds to sigma70 RNA polymerase holoenzyme to globally regulate gene expression in response to the shift from exponential growth to stationary phase. We have computationally identified >100 new 6S RNA homologs in diverse eubacterial lineages. Two abundant Bacillus subtilis RNAs of unknown function (BsrA and BsrB) and cyanobacterial 6Sa RNAs are now recognized as 6S homologs. Structural probing of E. coli 6S RNA and a B. subtilis homolog supports a common secondary structure derived from comparative sequence analysis. The conserved features of 6S RNA suggest that it binds RNA polymerase by mimicking the structure of DNA template in an open promoter complex. Interestingly, the two B. subtilis 6S RNAs are discoordinately expressed during growth, and many proteobacterial 6S RNAs could be cotranscribed with downstream homologs of the E. coli ygfA gene encoding a putative methenyltetrahydrofolate synthetase. The prevalence and robust expression of 6S RNAs emphasize their critical role in bacterial adaptation.     

Interaction of RNA with transformed glucocorticoid receptor. II. Identification of the RNA as transfer RNA

An endogenous RNA (designated as PIVB RNA), which is capable of associating with the 4 S glucocorticoid receptor (GR) to generate the 6 S form, has been purified from AtT-20 cells (Ali, M., and Vedeckis, W. V. (1987) J. Biol. Chem., 262, 6771-6777). We describe here the physiochemical properties, GR-RNA interaction characteristics, and the chemical identification of PIVB RNA. 32P-Labeled PIVB RNA was similar to transfer RNA (tRNA) in its sedimentation coefficient (4 S) on sucrose gradients, electrophoretic mobility on formaldehyde-agarose gels, and receptor binding characteristics. The amino acid acceptor activity of PIVB RNA displayed a typical tRNA-dependent saturation curve and was 2-3-fold higher than that of homologous rabbit liver tRNA when tested using rabbit liver aminoacyl-tRNA synthetase. The purified [3H] aminoacyl-PIVB complex was also capable of binding to the 4 S GR to generate the 6 S form. The analysis of PIVB RNA on an acrylamide-urea sequencing gel revealed that it contained a major tRNA of 76 nucleotides and other minor tRNA species of 74 and 78 nucleotides. The identity of the tRNA present in the PIVB RNA was indirectly deduced by analyzing the 3H-amino acids, liberated from the [3H]aminoacyl-PIVB RNA (tRNA) complex, and subsequent analysis on an amino acid analyzer. PIVB RNA mainly contained tRNAArg (51.8%), tRNALys (17.1%), and tRNAHis (9.2%) which together accounted for 78% of the total PIVB tRNA. The remaining 22% of tRNA was contributed by threonine, valine, aspartic acid, alanine, and phenylalanine tRNAs. The GR displayed no species specificity, and tRNA samples from mouse, cow, rabbit, yeast, and Escherichia coli can bind to the mouse 4 S GR to generate the 6 S form. However, PIVB RNA did not affect the sedimentation profiles of albumin, chymotrypsinogen, and histone, indicating that PIVB RNA does not bind to all proteins. Thus, there may exist some specificity both at the level of protein (GR) and the selection of RNA (tRNA). The GR binding to PIVB RNA occurred at low (nM) receptor concentration, and PIVB RNA showed limited capacity to shift 4 S GR to the 6 S form. 22.4 X 10(-11) mol of PIVB RNA can completely shift 4.8 X 10(-13) mol of 4 S GR to 6 S. That is, PIVB RNA has to be in a 500-600-fold excess over the amounts of GR to observe a stable 6 S GR X RNA complex on sucrose gradients. These results conclusively demonstrate that the transformed GR specifically binds to endogenous tRNA.     

Interaction of RNA with transformed glucocorticoid receptor. I. Isolation and purification of the RNA

The glucocorticoid receptor (GR) from mouse AtT-20 pituitary tumor cells, when transformed using a variety of in vitro protocols, yields a DNA-binding RNA-containing 6 S form. In order to better understand the physiological role of RNA interaction with the transformed GR, we have isolated and purified the putative RNA from AtT-20 cells. [3H]Triamcinolone acetonide-labeled cytosolic GR was transformed, using Sephadex G-25 filtration, to yield the RNA-containing 6 S GR. The transformed 6 S GR was separated on DEAE-cellulose into the 4 S GR (eluting at about 100 mM KCl) while its associated RNA eluted at 0.30-0.45 M KCl. The addition of only these RNA fractions to the 4 S GR can reconstitute 6 S GR as shown on 5-20% sucrose gradients. RNA (0.3-0.45 M KCl fractions) was further purified by hydroxylapatite chromatography, and the bound RNA (eluted at approximately 70 mM PO4(-2)) was then loaded onto preparative 5-20% sucrose gradients to separate RNA on the basis of size (sedimentation rate). A uniform class of RNA sedimenting at 4 S was obtained and then adsorbed to oligo(dT)-cellulose columns. The unbound fraction (poly(A-)) was capable of shifting 4 S GR to 6 S. Using these chromatographic procedures about 90% of the cellular RNA, incapable of reconstituting the 6 S GR from the 4 S form, was eliminated. The 4 S GR was covalently cross-linked with the purified RNA (termed PIVB RNA) using formaldehyde. The resulting cross-linked GR X RNA complexes were shown to sediment at the density of ribonucleoprotein (1.38 g/cm3) in CsCl gradients and at the 6 S position in high salt sucrose gradients. The hydrolysis of PIVB RNA with ribonuclease A prevented the formation of high salt-resistant ribonucleoprotein complexes, indicating that the GR may be in close contact with PIVB RNA. Electrophoresis of the PIVB RNA on 5% agarose-formaldehyde-denaturing gels yielded one major band with a molecular size of approximately 75 bases. It thus appears that an endogenous 4 S RNA (PIVB RNA) of about 25 kDa specifically interacts with the monomeric 4 S GR to yield the 6 S GR.     

Characterization of bovine viral diarrhea virus RNA.

RNA extracted from isopycnically banded [3-H]uridine-labeled bovine viral diarrhea virus with sodium dodecyl sulfate was resolved into one major and two minor components by both sedimentation analysis and electrophoresis in polyacrylamide gels. The major RNA component was estimated to have a 38S sedimentation coefficient. The minor RNA components were estimated to have S values of 31 and 24. The approximate colecular weights were calculated to be 3.22 times 10-6 (38S), 2.09 times 10-6 (31S), and 1.22 times 10-6 (24S). A single broad peak of radioactivity, maximum at 24S, was obtained when sedimentation was conducted under conditions of low ionic strength. All three RNA components were found to be susceptible to digestion with RNase. The presence of multiple RNA components in heterogeneous populations of infectious virus is discussed.     

RNA of mouse hepatitis virus.

The RNA of mouse hepatitis virus, a coronavirus, was isolated from the virus released early in the infection and analyzed by sucrose gradient sedimentation and electrophoresis. It was found to consist of a piece of single-stranded RNA of about 60S. Its molecular weight was estimated to be 5.4 X 10(6) by electrophoresis in methylmercury-agarose gels. At least one third of the RNA contained polyadenylated sequences. It is, therefore, probably positive stranded. The virus harvested late in the infection contained, in addition to 60S, some 30 to 50S RNA that are possibly degradation products of the 60S RNA. No difference in the electrophoretic behavior could be detected between the RNA isolated from a pathogenic (JHM) and a nonpathogenic (A59) strain.     

Gel Electrophoresis of Avian Leukosis and Sarcoma Viral RNA in Formamide: Comparison with Other Viral and Cellular RNA Species

Class a and class b 30 to 40S RNA subunits obtained by heat dissociation from the 60 to 70S RNA complex of avian tumor viruses were compared with several RNA standards by electrophoresis in formamide-polyacrylamide gels. Class a RNA was found to have a lower electrophoretic mobility and hence probably a higher molecular weight than class b RNA. The absolute molecular weight of class a and b RNA could not be determined with accuracy, because the relationship between logarithm of molecular weight and mobility of the RNA standards was not linear. The size of class a RNA fell into the range of 2.4 x 10(6) to 3.4 x 10(6) daltons and that of class b into the range of 2.2 x 10(6) to 2.9 x 10(6) daltons, depending on the standards used. The possible biological significance of this difference is discussed.     

Metabolism of pre-messenger RNA splicing cofactors: modification of U6 RNA is dependent on its interaction with U4 RNA.

The requirements for the formation of pseudouridine (psi) in U4 and U6 RNAs, cofactors in the splicing of pre-messenger RNA, were investigated in vitro using HeLa nuclear (NE) and cytoplasmic (S100) extracts. Maximal psi formation for both RNAs was extract order-dependent. Maximal psi formation in U4 RNA required incubation in S100 followed by the addition of NE, paralleling the in vivo maturation pathway of U4 RNA. In contrast, maximal formation of psi in U6 RNA required incubation in NE followed by the addition of S100 extract. Since U6 RNA does not exit the nucleus in vivo the contribution of S100 was investigated. In experiments where the extracts were treated with micrococcal nuclease to digest endogenous snRNAs, the efficient formation of psi in U6 RNA was dependent on the presence of U4 RNA, but not in U5 RNA or tRNA. When mutant U4 RNAs that inhibit or strengthen the interaction between U4 RNA, and U6 RNA were substituted for wild-type U4 RNA, the results confirmed the need for the interaction between these two RNAs for psi formation in U6 RNA. U6 RNA isolated from glycerol gradients after incubation in extracts had four times as much psi when associated with U4 RNA.     

Characterization of RNA from equine infectious anemia virus.

The genome of equine infectious anemia virus, a nononcogenic retrovirus, has been characterized by velocity sedimentation, electrophoresis in polyacrylamide gels, buoyant density in CS2SO4, and susceptibility to nuclease digestion. The nucleic acid of purified virus was resolved by sedimentation analysis into a fast-sedimenting genome component, which comprises about two-thirds of the virion RNA, and a slow-sedimenting RNA, which is probably comprised of host-derived tRNA and a trace amount of 5S RNA. The fast-sedimenting RNA had a sedimentation coefficient of 62S and a molecular weight of 5.4 X 10(6) to 5.6 X 10(6), as determined by sedimentation velocity and electrophoretic mobility. Upon heat denaturation, [3H]uridine-labeled 62S RNA dissociated into material comprised of 90 to 95% single-stranded species, sedimenting predominantly at 34S, with a molecular weight of 2.7 X 10(6) to 2.9 X 10(6) and 5 to 10% 4S RNA. The 62S RNA was predominantly single-stranded but contained double-stranded regions, as indicated by partial resistance to RNase IA and SI nuclease and by a lower buoyant density in CS2SO4 than that of the single-stranded 34S RNA derived by heat denaturation. These data indicated that the viral genome consisted of two 34S subunits of single-stranded RNA held in a high-molecular-weight complex with 4S RNA by a mechanism involving a small degree of base pairing. Thus, the structure of equine infectious anemia virus RNA is similar to that of other retroviruses.     

A new class of small nuclear RNA molecules synthesized by a type I RNA polymerase in HeLa cells

A new class of previously undetected small RNA molecules with a range of discrete sizes between 6S and 10S has been identified in HeLa cell nuclei. They differ in size and location from the previously described small nuclear RNA species (snRNA). These RNA molecules were initially found by selective RNA labeling in vitro in isolated nuclei. The in vitro products migrate in gel electrophoresis in the region from 6–10S with predominant components between 8S and 10S. They are labeled in the presence of very high concentrations of α-amanitin (150–400 μg/ml), suggesting they are synthesized by a type I polymerase. Unlike the major polymerase I product, ribosomal precursor RNA, however, these molecules are found in the nucleoplasm and their labeling is not affected by pretreatment of cells with low concentrations of actinomycin D (0.04 μg/ml). Their formation by a presumptive polymerase I type of enzyme is the basis of their tentative designation as small nuclear polymerase I (snPI) RNAs.The snPI RNA molecules appear to be associated with chromatin and the nuclear matrix. They can be selectively eluted from nuclei leaving most of hnRNA behind. This association is used as the basis of fractionation procedures which separate these molecules from hnRNA and permit the demonstration of the synthesis of at least the most predominant of these RNA molecules in vivo. w