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At least 217 records · Page 12Linked to original sources
PubMed1994

A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli.

We have determined that 10Sa RNA (one of the small stable RNAs found in Escherichia coli) has an interesting structural feature: the 5' end and the 3' end of 10Sa RNA can be arranged in a structure that is equivalent to a half-molecule (acceptor stem and TFC stem-loop) of alanine tRNA of E. coli. Primer-extension analysis of 10Sa RNA extracted from a bacterial mutant with temperature-sensitive RNase P function revealed that the precursor to 10Sa RNA (pre-10Sa RNA) is folded into a pre-tRNA-like structure in vivo such that it can be cleaved by RNase P to generate the 5' end of the mature 10Sa RNA. The purified 10Sa RNA can be charged with alanine in vitro. Disruption of the gene encoding 10Sa RNA (ssrA) caused a reduction in the rate of cell growth, which was especially apparent at 45 degrees C, and a reduction in motility on semisolid agar. These phenotypic characteristics of the deletion strain (delta ssrA) allowed us to investigate the effects of some mutations in 10Sa RNA in vivo, although the exact function of 10Sa RNA still remains unclear. When the G.U pair (G3.U357) in 10Sa RNA, which may be equivalent to the determinant G.U pair of alanine tRNA, was changed to a G.A or G.C pair, the ability to complement the phenotypic mutations of the delta ssrA strain was lost. Furthermore, this inability to complement the mutant phenotypes that was caused by the substitution of the determinant bases by a G.A pair could be overcome by the introduction of a gene encoding alanyl-tRNA synthetase (alaS) on a multicopy plasmid. The evidence suggests that the proposed structural features of 10Sa RNA are indeed manifested in vivo.

Base Sequence↗
PubMed1994

The Epstein-Barr virus (EBV) small RNA EBER1 binds and relocalizes ribosomal protein L22 in EBV-infected human B lymphocytes.

Epstein-Barr virus (EBV), an oncogenic herpesvirus, encodes two small RNAs (EBERs) that are expressed at high levels during latent transformation of human B lymphocytes. Here we report that a 15-kDa cellular protein called EAP (for EBER associated protein), previously shown to bind EBER1, is in fact the ribosomal protein L22. Approximately half of the L22 in EBV-positive cells is contained within the EBER1 ribonucleoprotein (RNP) particle, whereas the other half residues in monoribosomes and polysomes. Immunofluorescence with anti-L22 antibodies demonstrates that L22 is localized in the cytoplasm and the nucleoli of uninfected human cells, as expected, whereas EBV-positive lymphocytes also show strong nucleoplasmic staining. In situ hybridization indicates that the EBER RNPs are predominantly nucleoplasmic, suggesting that L22 relocalization correlates with binding to EBER1 in vivo. Since incubation of uninfected cell extracts with excess EBER1 RNA does not remove L22 from preexisting ribosomes, in vivo binding of L22 by EBER1 may precede ribosome assembly. The gene encoding L22 has recently been identified as the target of a chromosomal translocation in certain patients with leukemia, suggesting that L22 levels may be a determinant in cell transformation.

B-Lymphocytes↗
PubMed2006

Aptamer to ribozyme: the intrinsic catalytic potential of a small RNA.

The discovery of RNA-based catalysis 23 years ago dramatically changed the way biologists and biochemists thought of RNA. In the recent past, several ribozymes structures have provided some answers as to how catalysis is accomplished and how it relates to RNA structure and folding. However, there is still little information as to how catalytic activity evolved. Here we show that the small malachite green-binding aptamer has intrinsic catalytic potential that can be realized by designing the proper substrate. The charge distribution within the RNA binding pocket stabilizes the transition state of an ester hydrolysis reaction and thus accelerates the overall reaction. The results suggest that electrostatic forces can contribute significantly to RNA-based catalysis. Moreover, even simple RNA structures that have not been selected for catalytic properties can have a basic catalytic potential if they encounter the right substrate. This provides a possible starting point for the molecular evolution of more complex ribozymes.

Aptamers, Nucleotide↗
PubMed1995

Determination and comparative analysis of the small RNA genomic sequences of California encephalitis, Jamestown Canyon, Jerry Slough, Melao, Keystone and Trivittatus viruses (Bunyaviridae, genus Bunyavirus, California serogroup).

The nucleotide sequences of the small (S) genomic RNAs of six California (CAL) serogroup bunyaviruses (Bunyaviridae: genus Bunyavirus) were determined. The S RNAs of two California encephalitis virus strains, two Jamestown Canyon virus strains, Jerry Slough virus, Melao virus, Keystone virus and Trivittatus virus contained the overlapping nucleocapsid (N) and non-structural (NSs) protein open reading frames (ORFs) as described previously for the S RNAs of other CAL serogroup viruses. All N protein ORFs were 708 nucleotides in length and encoded a putative 235 amino acid gene product. The NSs ORFs were found to be of two lengths, 279 and 294 nucleotides, which potentially encode 92 and 97 amino acid proteins, respectively. The complementary termini and a purine-rich sequence in the 3' non-coding region (genome-complementary sense) were highly conserved amongst CAL serogroup bunyavirus S RNAs. Phylogenetic analyses of N ORF sequences indicate that the CAL serogroup bunyaviruses can be divided into three monophyletic lineages corresponding to three of the complexes previously derived by serological classification. The truncated version of the NSs protein, which is found in five CAL serogroup bunyaviruses, appears to have arisen twice during virus evolution.

Amino Acid Sequence↗
PubMedDate not supplied

A bioinformatics based approach to discover small RNA genes in the Escherichia coli genome.

The recent explosion in available bacterial genome sequences has initiated the need to improve an ability to annotate important sequence and structural elements in a fast, efficient and accurate manner. In particular, small non-coding RNAs (sRNAs) have been difficult to predict. The sRNAs play an important number of structural, catalytic and regulatory roles in the cell. Although a few groups have recently published prediction methods for annotating sRNAs in bacterial genome, much remains to be done in this field. Toward the goal of developing an efficient method for predicting unknown sRNA genes in the completed Escherichia coli genome, we adopted a bioinformatics approach to search for DNA regions that contain a sigma70 promoter within a short distance of a rho-independent terminator. Among a total of 227 candidate sRNA genes initially identified, 32 were previously described sRNAs, orphan tRNAs, and partial tRNA and rRNA operons. Fifty-one are mRNAs genes encoding annotated extremely small open reading frames (ORFs) following an acceptable ribosome binding site. One hundred forty-four are potentially novel non-translatable sRNA genes. Using total RNA isolated from E. coli MG1655 cells grown under four different conditions, we verified transcripts of some of the genes by Northern hybridization. Here we summarize our data and discuss the rules and advantages/disadvantages of using this approach in annotating sRNA genes on bacterial genomes.

Base Sequence↗
PubMed1994

Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit.

It is now generally accepted that 16S and 23S ribosomal RNA play important roles in the decoding and peptidyl transferase activities of ribosomes. Despite their complex structures and numerous associated proteins it is possible that small domains of these rRNAs can fold and function autonomously, particularly those that appear devoid of protein interactions. One candidate for such a domain is the decoding region, located near the 3' end of 16S rRNA (Fig. 1a, b). Consistent with this hypothesis, aminoglycoside antibiotics that interact with the decoding region in 30S subunits interact with other RNAs in the absence of proteins. In addition, certain activities of self-splicing introns, at least superficially, resemble translational decoding. We report here that an oligoribonucleotide analogue of the decoding region interacts with both antibiotic and RNA ligands of the 30S subunit in a manner that correlates with normal subunit function. The activities of the decoding region analogue suggest that the intimidating structural complexity of the ribosome can be, to some degree, circumvented.

Anti-Bacterial Agents↗
PubMed2001

Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule.

A novel growth phase-associated two-component-type regulator, Fas (fibronectin/fibrinogen binding/haemolytic activity/streptokinase regulator), of Streptococcus pyogenes was identified in the M1 genome sequence, based on homologies to the histidine protein kinase (HPK) and response regulator (RR) part of the Staphylococcus aureus Agr and Streptococcus pneumoniae Com quorum-sensing systems. The fas operon, present in all 12 tested M serotypes, was transcribed as polycystronic message (fasBCA) and contained genes encoding two potential HPKs (FasB and FasC) and one RR (FasA). Downstream of fasBCA, we identified a small 300 nucleotide monocistronic transcript, designated fasX, that did not appear to encode true peptide sequences. Measurements of luciferase promoter fusions revealed a growth phase-associated transcription of fasBCA and fasX, with peak activities during the late exponential phase. Insertional mutagenesis disrupting fasBCA and fasA led to a phenotype similar to agr-null mutations in S. aureus, with prolonged expression of extracellular matrix protein-binding adhesins and reduced expression of secreted virulence factors such as streptokinase and streptolysin S. In addition, fasX transcription was dependent on the RR FasA; however, deletion mutagenesis of fasX resulted in a similar phenotype to that of the fasBCA or fasA mutants. Complementation of the fasX deletion mutant, with the fasX gene expressed in trans from a plasmid, restored the wild-type fasBCA regulation pattern. This strongly suggested that fasX, a putative non-translated RNA, is the main effector molecule of the fas regulon. However, using spent culture supernatants from wild-type and fas mutant strains, we were not able to show an influence on the logarithmic growth phase expression of fas and dependent genes. Thus, despite structural and functional similarities between fas and agr, to date the fas operon appears not to be involved in group A streptococcal (GAS) quorum-sensing regulation.

Amino Acid Sequence↗
PubMed1993

Amplification of polyadenylated nontranslated small RNA sequences (POLADS) during superinfection correlates with the inhibition of viral and cellular protein synthesis.

The production and the role of POLADS in cells infected with vaccinia virus (VV) was studied in doubly infected HeLa and L cells. In cells first infected with VV followed by superinfection with UV-irradiated virus (VVuv), referred to as VV + VVuv, the course of viral polypeptide synthesis was not significantly altered. However, if cells were first infected with VVuv, followed by superinfection with VV, referred to as VVuv + VV, both host and viral polypeptide synthesis was compromised. Labeling of such infected cells with 3H-adenosine revealed that infection with VVuv or VVuv + VV, caused an amplification of incorporation of label both in the large and small size class RNAs compared to RNAs obtained from a normal infection. On the other hand, labeling of cells with 3H-adenosine which were infected with VV + VVuv, caused no significant change in the labeling pattern of large and small size class RNAs compared to labeled RNAs from normal infection. The large size class RNAs isolated from infection with VVuv or VVuv + VV, were translated in the reticulocyte lysate cell-free system much more productively compared to the same size RNAs obtained from normal infection. When these large size class RNAs were added together with HeLa cell mRNAs to the cell-free translational system, competition between the two types of mRNAs ensued. The small size class RNAs (POLADS) isolated from infection with VVuv or VVuv + VV, had little translational activity, but when added together with HeLa cell mRNAs, caused a striking inhibition of HeLa cell mRNA translation which was more pronounced than the inhibition caused by the small size class RNAs obtained from normal infections. POLADS obtained from infection with VV or VV + VVuv inhibited HeLa cell protein synthesis to the same extent and were about two times less active than the POLADS obtained from infection with VVuv or VVuv + VV. These results demonstrate that if cells are first infected with normal virus, the superinfecting UV-irradiated virus has little or no effect on the course of VV replication, suggesting that the production of POLADS under these conditions is regulated. However, when cells are first infected with VVuv, POLADS production is amplified to an "abnormal" level, thus, both viral polypeptide synthesis and replication of the superinfecting VV is compromised resulting in lower yields of virus.

Animals↗
PubMedDate not supplied

Expression of enhanced levels of small RNA polymerase III transcripts encoded by the B2 repeats in simian virus 40-transformed mouse cells.

Although specific viral genes are known which are sufficient to transform certain types of cells, viral transformation has been shown to be accompanied by a change in the level of expression of cellular genes. Assuming that this latter class of genes is involved in some way in the establishment of the transformed state, we and others have sought to characterize them. To this end, we constructed a complementary DNA library from a simian virus 40 (SV40)-transformed mouse cell line and screened this library for induced genes. Here we focus on the characterization of a specific cDNA clone called 10 lambda 5. We show that the clone 10 lambda 5 contains a B2 repeat, and that the levels of the small heterogeneously sized B2 cytoplasmic RNAs homologous to 10 lambda 5 are enhanced in transformed cells. Our studies show that these RNAs are a specific class of polymerase III transcripts containing a highly conserved 5' end. SV40 transformation results, therefore, in the activation of specific polymerase III transcripts as well as specific polymerase II transcripts.

Animals↗
PubMed1993

Structure of a small RNA hairpin.

The hairpin stem-loop form of the RNA oligonucleotide rCGC(UUU)GCG has been studied by NMR spectroscopy. In 10 mM phosphate buffer this RNA molecule forms a unimolecular hairpin with a stem of three base pairs and a loop of three uridines, as judged by both NMR and UV absorbance melting behavior. Distance and torsion angle restraints were determined using homonuclear proton-proton and heteronuclear proton-phosphorus 2-D NMR. These values were used in restrained molecular dynamics to determine the structure of the hairpin. The stem has characteristics of A-form geometry, although distortion from A-form occurs in the 3'-side of the stem, presumably to aid in accommodating the small loop. The loop nucleotides adopt C2'-endo conformations. NOE's strongly suggest stacking of the uracils with the stem, especially the first uracil on the 5'-side of the loop. The reversal of the chain direction in the loop seems to occur between U5 and U6. Loop structures produced by molecular dynamics simulations had a wide range of conformations and did not show stacking of the uracils. A flexible loop with significant dynamics is consistent with all the data.

Base Sequence↗
PubMed2006

Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition.

Riboswitches are noncoding mRNA elements that bind small-molecule metabolites with high affinity and specificity, and they regulate the expression of associated genes. The thi-box riboswitch can exhibit a 1000-fold higher affinity for thiamine pyrophosphate over closely related noncognate compounds such as thiamine monophosphate. To understand the chemical basis of thi-box pyrophosphate specificity, we have determined crystal structures of an E. coli thi-box bound to thiamine pyrophosphate, thiamine monophosphate, and the structural analogs benfotiamine and pyrithiamine. When bound to monophosphorylated compounds, the RNA elements that recognize the thiamine and phosphate moieties of the ligand move closer together. This allows the riboswitch to recognize the monophosphate in a manner similar to how it recognizes the beta-phosphate of thiamine pyrophosphate. In the pyrithiamine complex, the pyrophosphate binding site is largely unstructured. These results show how the riboswitch can bind to various metabolites, and why the thi-box preferentially binds thiamine pyrophosphate.

Crystallography, X-Ray↗
PubMed1986

Detection by ELISA of antibodies to small RNA protein particles in systemic lupus erythematosus patients whose sera lack precipitins.

Three hundred five consecutive patients with systemic lupus erythematosus (SLE) have been evaluated for antibodies to the RNA protein particles, Sm, nRNP, Ro/SSA, and La/SSB. Precipitating antibodies were found in 224 (73.1%). With the use of ELISA methodology with affinity-purified antigens, 76 sera from precipitin-negative patients were assayed and 35 had elevated levels of one or more of the autoantibodies. Of these, 27 (36%) had elevated anti-nRNP and 17 (22%) had elevated anti-Ro/SSA. Six of the seven patients with anti-La/SSB (18%) also had anti-Ro/SSA paralleling the close association of these two antibodies noted by precipitin analysis in which 38 of 39 patients with anti-La/SSB precipitins also have anti-Ro/SSA precipitins. Thus, 258 of the 305 SLE sera (84.6%) have elevated anti-RNA protein levels. This represents a minimum estimate of the frequency of these antibodies since longitudinal study of individual patients shows that anti-RNA protein levels may vary from normal to 1000 times over background at different times. Quantitative studies over time in two patients with both anti-Sm and anti-nRNP show that anti-nRNP increased 10- to 100-fold while anti-Sm changed little. These quantitative changes may relate to clinical phenomena.

Adolescent↗
PubMed1983

Post-transcriptional control of expression of the repA gene of plasmid R1 mediated by a small RNA molecule.

Fusions between the repA gene of plasmid R1 (required for autonomous plasmid replication) and the lac genes have been the basis for in vivo studies of regulation of repA expression. Two gene products--the CopA RNA and the CopB protein--act as inhibitors of repA expression. Comparison of the effects of addition in trans of the two Cop functions on transcription and translation of repA-lac gene fusions show that the CopB protein represses transcription of the repA gene, whereas the CopA RNA interferes with the RepA mRNA in such a way that effective translation is inhibited. The CopA RNA does not seem to have a direct effect on the transcription of the repA gene but, as a consequence of the posttranscriptional regulation, transcriptional polarity within the repA gene is observed. It is also shown that the CopA RNA interacts with its target (CopT) only when the region is transcribed to form RepA mRNA.

Artificial Gene Fusion↗
PubMed2002

In vivo effects of the Epstein-Barr virus small RNA EBER-1 on protein synthesis and cell growth regulation.

Recent studies have suggested a role for the Epstein-Barr virus-encoded RNA EBER-1 in malignant transformation. EBER-1 inhibits the activity of the protein kinase PKR, an inhibitor of protein synthesis with tumour suppressor properties. In human 293 cells and murine embryonic fibroblasts, transient expression of EBER-1 promoted total protein synthesis and enhanced the expression of cotransfected reporter genes. However reporter gene expression was stimulated equally well in cells from control and PKR knockout mice. NIH 3T3 cells stably expressing EBER-1 exhibited a greatly increased frequency of colony formation in soft agar, and protein synthesis in these cells was relatively resistant to inhibition by the calcium ionophore A23187. Nevertheless clones containing a high concentration of EBER-1 were not invariably tumourigenic. We conclude that EBER-1 can enhance protein synthesis by a PKR-independent mechanism and that, although this RNA may contribute to the oncogenic potential of Epstein-Barr virus, its expression is not always sufficient for malignant transformation.

Animals↗
PubMed1991

10Sa RNA, a small stable RNA of Escherichia coli, is functional.

The ssrA gene, coding for the metabolically stable 10Sa RNA, affects cell growth. A mutant in which the chromosomal 10Sa RNA gene is interrupted by a cat insert does not produce detectable levels of 10Sa RNA, and it grows more slowly than the parental strain.

Blotting, Northern↗