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PubMed1993

Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure.

Regions of Escherichia coli ribonuclease P (RNase P) RNA in proximity to a bound transfer RNA (tRNA) substrate were mapped by photoaffinity. A photoaffinity cross-linking reagent was introduced at specific sites in the interior of the native tRNA structure by modification of the 5' ends of circularly permuted tRNAs (cptRNAs). The polymerase chain reaction was used for the production of cptRNA templates. After the amplification of a segment of a tandemly duplicated tRNA gene, the cptRNA gene was transcribed in vitro to produce cptRNA. Modified cptRNAs were cross-linked to RNase P RNA, and the conjugation sites in RNase P RNA were determined by primer extension. These sites occur in phylogenetically conserved structures and sequences and identify regions of the ribozyme that form part of the tRNA binding site. The use of circularly permuted molecules to position specific modifications is applicable to the study of many inter- and intramolecular interactions.

Affinity Labels↗
PubMed1977

Alteration of 5S RNA conformation by ribosomal proteins L18 and L25.

The effects of ribosomal proteins L18, L25 and L5 on the conformation of 5S RNA have been studied by circular dichroism and temperature dependent ultraviolet absorbance. The circular dichroism spectrum of native 5S RNA is characterized in the near ultraviolet by a large positive band at 267 nm and a small negative band at 298 nm. The greatest perturbation in the spectrum was produced by protein L18 which induced a 20% increase in the 267 nm band and no change in the 298 nm band. By contrast, protein L25 caused a small decrease in both bands. No effect was observed with protein L5. Simultaneous binding of proteins L18 and L25 resulted in CD changes equivalent to the sum of their independent effects. The UV absorbance thermal denaturation profile of the 5S RNA L18 complex lacked the pre-melting behavior characteristic of 5S RNA. Protein L25 had no effect on the 5S RNA melting profile. We concluded that protein L18 increases the secondary, and possible the tertiary structure of 5S RNA, and exerts a minor stabilizing effect on its conformation while protein L25 causes a small decrease in 5S RNA secondary structure. The implications of these findings for ribosome assembly and function are discussed.

Chemical Phenomena↗
PubMed2002

Descent of a split RNA.

tmRNA is found in the usual one-piece form in most cyanobacteria, but a small clade has an unusual two-piece form, resulting from processing of a circularly permuted precursor RNA. Here, the secondary structure of the cyanobacterial one-piece tmRNA is established by phylogenetic sequence comparison; it deviates from most tmRNAs in having an extra (fifth) pseudoknot and a branched structure at the end of the tag reading frame. Patterns of sequence conservation between the two cyanobacterial tmRNA types suggest particular events in the evolution of the two-piece form. A simple gene permutation model of tandem duplication followed by loss of the outermost segments appears inadequate. An additional rearrangement is proposed, in which a structure impaired by deletion was regenerated at the expense of a neighboring structure.

Base Sequence↗
PubMed1997

Identification of a promoter region generating Sry circular transcripts both in germ cells from male adult mice and in male mouse embryonal gonads.

The mouse testis determining gene Sry is expressed in somatic cells of the differentiating male gonad as a linear transcript, encoding a transcription factor containing an HMG box. In the adult mouse testis, Sry expression occurs in meiotic and postmeiotic germ cells. The mouse genomic Sry locus is characterized by two arms of a large inverted repeat, flanking a unique region that, between an acceptor and a donor splice site, contains a single exon encoding the Sry protein. In germ cells from the adult mouse testis, Sry RNA is a circular molecule, which is generated by an inverted splicing event that utilizes the above-mentioned splice sites. Thus, a circular exon is spliced out starting from a large linear RNA precursor containing both arms of the inverted repeat, which pair and generate a large stem-loop structure. Using reverse transcription-polymerase chain reaction and an RNase protection assay, we have now mapped the 5' end of this precursor RNA in the 5' arm of the inverted repeat. Gel mobility shift assay and in vitro transcription with nuclear extracts from adult germ cells further confirm that a region immediately 5' upstream of two transcriptional initiation sites of the precursor RNA contains a promoter sequence in which two consensus Sry binding sequences are specifically recognized by nuclear factors present in adult germ cells but not in Sertoli cells. We also show that the linear precursor of the Sry circular transcript and its splicing product are specifically expressed not only in adult germ cells but also in male embryonal gonads between 11.5 and 13.5 days postcoitum, immediately after the expression of the linear transcript starting from the unique region.

Animals↗
PubMed2005

Mechanistic insights on the folding of a large ribozyme during transcription.

RNA folding during transcription resembles folding in a cellular environment. We previously investigated the folding of a large ribozyme derived from a bacterial RNase P RNA during its transcription by the Escherichia coli RNA polymerase and the effect of the elongation factor NusA. We found that transcriptional pausing at a specific site induced by NusA significantly altered the folding pathway. In this work, we compare folding during transcription by the E. coli RNA polymerase of circularly permuted variants and site-specific mutants of the RNase P ribozyme to elucidate the molecular mechanism of transcriptional pausing and RNA folding. The effect of NusA-induced pausing depends on the order of RNA synthesis and only affects local folding of the RNA. Pausing likely prevents a misfolded structure between the 5' strand of a helix and its adjacent junction located in the specificity domain and a region known to bind single-stranded RNA located in the catalytic domain. These results lead to a structural model on how transcriptional pausing affects folding of RNase P RNA. Structural rearrangements of a nascent RNA transcript enhanced by transcriptional pausing may be a general feature of RNA folding during transcription.

Base Sequence↗
PubMed1975

T7 RNA polymerase: conformation, functional groups, and promotor binding.

Circular dichroic spectra of T7 RNA polymerase show minima at 222 nm ([theta]m=-7.9 X 10(3) deg cm2/dmol) and 208 nm ([theta]m =-7.55 X 10(3) deg cm2/dmol) and a maximum at 193 nm ([theta]m = 1.2 X 10(4) deg cm2/dmol). The small mean residue ellipticity above 200 nm indicates that the secondary structure contains approximately 12% alpha helix. The secondary structure is unaltered by high salt, glycerol, -SH reagents, nitration of tyrosyl residues, and chelating agents. Binding of the native enzyme to [32P]T7 DNA has been measured by the retention of the protein-[32P]DNA complexes on nitrocellulose filters. At 37degrees T7 RNA polymerase binds to its promoters in the absence of NTP's. Binding and catalytic activity are both abolished at 0degree. Binding of the initiating [gamma-32P]GTP can also be detected by the filter binding assay. Native T7 RNA polymerase is inactivated by reaction with 1 mol of 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or 1 mol of [14C]iodoacetamide. The latter reaction is blocked by Nbs2 suggesting that a single -SH group is required for activity. Alkylation of the -SH group does not alter binding of the enzyme to the DNA template, but modifies the binding of GTP to the enzyme. Nitration of approximately4 surface tyrosyl residues of the protein prevents binding to T7 DNA. The restriction endonuclease, Hpa II, cuts T7 DNA into approximately40 fragments and reduces total RNA synthesis by T7 RNA polymerase by 70%. Fragmentation of the DNA template by Hpa II does not alter the rate of RNA chain initiation by T7 polymerase, and restriction fragments accounting for approximately25% of the T7 DNA still bind tightly to the enzyme. Thus the T7 RNA polymerase promoters remain intact on the restriction fragments. Gel electrophoresis of the transcription products, using restriction fragments as templates, show that of the seven in vitro transcripts produced by T7 RNA polymerase from whole T7 DNA, only the smallest (representing the last 1.5% of the genome) is transcribed from Hpa II fragments. The remaining transcripts are replaced by six new and much shorter mRNA's. The DNA fragments containing the promoters for these mRNA's have been removed from the fragment mix by binding them to the enzyme and retaining the complexes on nitrocellulose filters.

Coliphages↗
PubMed1977

Search for virus specific DNA sequences and viral particles in mitochondria of avian leukemic myeloblasts.

The intracellular localization of the avian myeloblastosis virus (AMV) genome was studied. Nuclear and mitochondrial DNAs from myeloblasts were examined by hybridization with 32P labeled AMV-RNA of high molecular weight for the presence of virus specific DNA sequences. Nuclear DNA (nDNA) from myeloblasts specifically hybridized with viral RNA, whereas purified closed circular mitochondrial DNA (mtDNA) did not hybridize with viral RNA. It was therefore concluded that viral genome was present in nuclear DNA and not in mitochondrial DNA. Likewise, in normal chick cells, nDNA but not mtDNA hybridized with viral RNA.

Avian Leukosis Virus↗
PubMed1973

Effect of circularity and superhelicity on transcription from bacteriophagelambda DNA.

We studied RNA synthesis in vitro from closed-circular lambda DNA molecules with varying degrees of superhelicity. The four circular templates examined had 0, -50, -110, and -160 superhelical turns under the conditions of the transcription assay. We found that the total amount of RNA synthesis increases as the template acquires more negative superhelical turns. This increased transcription results from more frequent initiation of RNA chains. Transcription of circular DNA with no superhelical turns appears to mimic RNA synthesis in vivo more closely than transcription from either highly superhelical or linear DNA with regard to two criteria: preferential transcription of the region corresponding to early genes and sensitivity to repression by lambda cI protein. We suggest that the physical basis for the increased initiation of RNA chains from superhelical DNA is the fact that unwinding events are energetically favored on a DNA molecule with negative superhelical turns. Possible general mechanisms are: (a) RNA polymerase must unwind the DNA duplex as a prelude to initiation; (b) the DNA itself must assume a new conformation at the promoter site which requires an unwinding of the DNA duplex.

Centrifugation, Density Gradient↗
PubMed1984

Complex of fd gene 5 protein and double-stranded RNA.

We report the formation of complexes of the single-stranded DNA binding protein encoded by gene 5 of fd virus, with natural double-stranded RNAs. In the first direct visualization of a complex of the fd gene 5 protein with a double-stranded nucleic acid, we show by electron microscopy that the double-stranded RNA complex has a structure which is distinct from that of complexes with single-stranded DNA and is consistent with uniform coating of the exterior of the double-stranded RNA helix by the protein. Circular dichroism spectral data demonstrate that the RNA double helix in the complex is undisrupted, and that perturbation of the 228-nm circular dichroism assigned to protein tyrosines can occur in the absence of intercalation of nucleotide bases with protein aromatic residues. Our findings emphasize the potential importance of interaction with the sugar-phosphate polynucleotide backbone in binding of the fd gene 5 protein to nucleic acids.

Circular Dichroism↗
PubMed1993

Structure of the maize mitochondrial replicon RNA b and its relationship with other autonomously replicating RNA species.

The mitochondria of maize plants with S-type cytoplasm possess a family of single and double-stranded RNA molecules, termed RNA plasmids, that replicate in a DNA-independent manner typical of RNA viruses. We have determined the sequence of the smallest and most abundant member of this family, a single-stranded RNA termed RNA b. The 719 nucleotide sequence of RNA b lacks open reading frames of significant length. Probes complementary to the determined (+) strand sequence identified two larger RNA species of 2900 and > 4000 nucleotides in S-type maize mitochondria, the smaller of which corresponds to a previously identified member of the RNA plasmid family designated RNA a. These probes also unexpectedly identified a low abundance form of RNA b in N-type maize mitochondria. (+) strand probes identified corresponding (-) strand forms in S cytoplasm that likely represent components of replication intermediates. Primer extension experiments demonstrated that RNA b is an internally deleted form of the larger RNA plasmid, RNA a, and that it has a discrete and homogeneous 5' terminus. The solubility of RNA b in 2 M-LiCl as well as structural modelling studies indicated that it has a very high degree of secondary structure. Analysis of specific cleavage products generated by treatment of RNA b-oligonucleotide duplexes with RNase H indicated that the only identifiable forms of RNA b were linear molecules with relatively homogeneous 3' termini; no circular forms were detected. RNA b was found to share a 13 nucleotide sequence with the circular cadang-cadang viroid; 11 of these nucleotides fall within the central conserved region of viroid RNAs. In addition, RNA b shares certain structural similarities with phage Q beta and "variant" RNAs that serve as in vitro templates for the Q beta replicase. Collectively, our results indicate that RNA b is a "defective-interfering" member of an RNA family representing a new and apparently distinct class of RNA replicons.

Base Sequence↗
PubMed1996

Secondary structure probing of potato spindle tuber viroid (PSTVd) and sequence comparison with other small pathogenic RNA replicons provides evidence for central non-canonical base-pairs, large A-rich loops, and a terminal branch.

Using PCR and in vitro transcription, linear (non-circular) unit-length (+)strand RNA molecules of a lethal PSTVd variant were produced which are able to initiate typical disease symptoms when inoculated into tomato. Non-denaturing gel electrophoresis shows that these transcripts can adopt the same two conformations as circular PSTVd molecules, namely a fast migrating rod-like and a slowly migrating cruciform structure. The rod-like conformer of two end-labelled transcripts was probed with nucleases and dimethyl sulphate, revealing that in solution its right part is identical to computer prediction. In the left part, however, three unique features could be substantiated. (1) In the central region a UV-cross-linkable loop is closed and thus contains non-canonical base-pairs ("loop E structure"). (2) Three large "pre-melting loops" are present at 25 degrees to 37 degrees C. The structure of the leftmost one, which is A-rich and conserved in most viroids, correlates with pathogenicity. (3) Two small stem-loops instead of an unbranched structure are found at the left terminus. These hairpins can form in all "large" viroids (approximately 300 nucleotides or longer), thus placing the dodecamer conserved among these viroids, GGUUCCUGUGGU, within the upper helix and the branch junction. A large viroid from Iresine lacks one of these hairpins, whereas all "small" viroids (approximately 300 nucleotides or smaller) lack both. In several plant virus satellite RNAs and the newt satellite RNA, the motif GAUUU(U) and dodecamer remnants appear in an equivalent structure comprising two or three hairpins. Using lead- and terbium-induced cleavage of the RNA, metal binding sites were found, mostly in loops. Thus, probing of PSTVd RNA and comparison with other.

Base Composition↗
PubMed1981

Spectroscopic studies of Congo Red binding to RNA polymerase.

The azo dye Congo Red has a high affinity for nucleotide-binding enzymes. We have studied the binding of Congo Red to RNA polymerase by circular dichroism (CD) and difference absorption spectroscopy, steady-state kinetics, and nitrocellulose filter-binding. Induced CD shows that a large number of Congo Red molecules bind to the holoenzyme. CD also demonstrates that the core enzyme at low ionic strengths has a distinctive Congo Red binding site which is not present in the holoenzyme, nor in the core enzyme at higher ionic strengths or in the presence of poly(dT). CD studies indicate that Congo Red can readily displace double-stranded polynucleotides (T7 DNA or poly[d(A-T)] from RNA polymerase. Single-stranded DNA (poly(dT) and T7 DNA in open complexes) is not displaced from RNA polymerase except at high Congo Red concentrations. Both kinetics and nitrocellulose filter-binding measurements support this conclusion. Difference spectra indicate that the bound Congo Red molecules undergo stacking. We postulate that RNA polymerase binds Congo Red in a region with which a segment of DNA normally interacts, and that Congo Red is a potent inhibitor because the stacked dye has a polyanionic character.

Binding Sites↗
PubMed1976

Proviral DNA of Moloney leukemia virus: purification and visualization.

Closed-circular proviral DNA of Moloney leukemia virus has been purified from a 10(7) excess of cellular and mitochondrial DNA. The DNA can be visualized in the electron microscope and has the contour length of a molecule with a moleculecular weight of about 5.5 + 10(6). Electron microscopic observation of a hybrid between viral RNA and this circular DNA confirms the viral origin of this molecule.

DNA, Circular↗
PubMed1976

Circular dichroism studies of ethidium bromide binding to the isolated nucleolus.

Circular dichroism in the 300-360 nm region and fluorescence induced by intercaltating binding of ethidum bromide to both DNA and RNA components were studied in isolated HeLa nucleoli. Both DNA and RNA compoents contribute to the induced dichroic elliticity. Digestion of nucleoli by RNase or DNase shows that most of the induced ellipticity comes from the DNA component. In nucleoli with an RNA/DNA = 0.8/1.0 the RNA component gives only 20% of the total ellipticity when measured at an ethidium bromide/DNA = 0.25. Spectro-fluorometric titration shows that ethidium bromide intercalates mostly into DNA in nucleoli. Both circular dichroism and fluorescence studies indicate that both DNA and RNA components in isolated nucleoli are less accessible to intercalating binding by ethidium bromide when compared to purified nucleolar DNA, DNA in chromatin or purified ribosomal RNA. Circular dichroic measurements of intercalating binding of ethidium bromide to to nucleoli may be used to study changes in nucleoli under different physiological or pathological conditions.

Binding Sites↗
PubMed1983

An RNA ligase from wheat germ which participates in transfer RNA splicing in vitro.

Transfer RNA half-molecules are intermediates in the splicing of tRNA precursors containing intervening sequences. We have utilized yeast tRNA half-molecules to identify and partially purify an ATP-dependent RNA ligase activity from extracts of wheat germ. This activity can complement a yeast tRNA endonuclease in vitro to efficiently splice 10 different yeast tRNA precursors. The products of in vitro splicing are a covalently joined tRNA and a circular intervening sequence RNA. The internucleotide bond formed at the splice junction is a 2'-phosphomonoester, 3',5'-phosphodiester structure. The 2'-phosphate originates from the 2',3'-cyclic phosphate at the 3' terminus of the 5' half-tRNA. The phosphodiester phosphate is derived from the gamma-phosphate of ATP.

Phosphorus Radioisotopes↗
PubMed1988

Evidence for a single rolling circle in the replication of potato spindle tuber viroid.

We analyzed in vivo-labeled RNA to determine which of the two proposed rolling-circle models is more likely to depict the replication cycle of potato spindle tuber viroid. A key feature distinguishing the two models is the presence of a circular monomeric minus strand in one and not the other. Chromatography on cellulose CF11 was used to purify a fraction containing the replication intermediates free from single-stranded progeny. Heat denaturation followed by gel electrophoresis was used to seek possible circular templates-species required for rolling-circle replication to take place. Upon heating, a (32)P-labeled RNA was released. Limited nuclease digestion ("nicking") revealed that this was a unitlength circular RNA. Fingerprinting identified it as a plus strand. No circular minus strands were detected in this population or in nuclease-treated samples containing RNase T1-resistant cores of the replication complex. Thus, potato spindle tuber viroid appears to use an asymmetric pathway in which minus strands are synthesized by rolling-circle copying, but plus strands are not. More details of the replication pathways used by various viroid-like RNAs are needed and will help to establish the evolutionary relationships among these infectious agents.

Journal Article↗