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PubMed1987

Structure of the ColE1 rop protein at 1.7 A resolution.

Structural details of the Rop protein from plasmid ColE1 are presented, with a description of the X-ray crystal structure determination and refinement at a nominal resolution of 1.7 A. The 63 amino acid protein is a dimer. Each monomer consists almost entirely of two alpha helices, the whole molecule forming a highly regular four-alpha-helix bundle. This may be approximated by a four-stranded rope with a radius of 7.0 A, a left-handed helical twist and a pitch of 172.5 A. The packing constraints for this novel type of coiled-coil structure are given. The protein acts in the control of plasmid replication via regulation of an RNA-RNA interaction in a manner not yet understood in atomic detail.

Amino Acid Sequence↗
PubMed1989

Kinetics of complementary RNA-RNA interaction involved in plasmid ColE1 copy number control.

Binding of a small antisense RNA (RNA I) to the primer transcript (RNA II) of plasmid ColE1 inhibits formation of primer for DNA polymerase I-mediated plasmid replication. It is thought that RNA I and RNA II transiently interact via their single-stranded loop regions to form an unstable complex that subsequently converts into a more stable complex by hybridization. Rom (or Rop) protein enhances the inhibitory effect of RNA I on replication by enhancing the binding of the two RNAs. In this paper, we develop a model for the kinetics of the RNA I-RNA II binding reaction, estimate the rate constants, and provide a quantitative description of the effects of Rom protein. We show that the reaction kinetics are consistent with a stepwise binding model in which Rom protein binds to RNA I and RNA II, while the RNAs are held together in a transient complex. Mutations that replace C.G pairs by T.A pairs in the RNA loop regions and thus display weaker hydrogen bonding between the loop regions should be associated with an increased rate of dissociation for the unstable complex. Our model predicts that such destabilization of the loop interactions leads to a greater enhancement in the binding rate by Rom protein. The available data support this prediction.

Bacterial Proteins↗
PubMed1989

Genetic methods for identification and characterization of RNA-RNA and RNA-protein interactions.

Two general strategies using pseudorevertants have been useful in identifying RNA-RNA and RNA-protein interactions, direct synthesis of compensatory changes to test models or in vivo selection of suppressors. The most successful application of these approaches has been in the elucidation of RNA-RNA interactions, in a large part because the rules for RNA base pairing are, at least partially, understood. Beyond the initial identification of interactions, suppressors also provide important starting points for further analysis. In the case of an unknown gene product (i.e., the RNA16 gene product), the suppressor provides a route to cloning and analysis of the gene. In addition, the construction of a specific suppressor in a RNA molecule can provide a starting point for the dissection of the biogenesis and function of that RNA. This aspect is most important when the RNA in question normally provides an essential cellular role or is encoded by a multigene family. For example, the construction of a message-specific 16 S RNA in Escherichia coli should, in principle, allow for a genetic dissection of rRNA biogenesis and function.

Base Composition↗
PubMed1983

Characterization of the yeast mitochondrial locus necessary for tRNA biosynthesis: DNA sequence analysis and identification of a new transcript.

Most components necessary for the biosynthesis of mitochondrial tRNAs are coded by nuclear genes, but one mitochondrial locus other than the tRNA genes themselves is required to make functional tRNAs in the yeast Saccharomyces cerevisiae. DNA sequence analysis of this yeast mitochondrial tRNA synthesis locus is reported here. This region of mitochondrial DNA is almost exclusively A+T-rich DNA with one G+C-rich element. Despite the unusual structure of the DNA in this region, we have demonstrated that it codes for a heretofore unidentified mitochondrial transcript about 450 bases in length. Since this RNA is the only RNA encoded by the tRNA synthesis locus, it must be the active agent of the locus. This RNA could either act autonomously through RNA-RNA interactions or as part of an RNA-protein complex to effect tRNA biosynthesis.

Base Sequence↗
PubMed1993

The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.

lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction.

Animals↗
PubMed1995

Dissecting RNA-protein interactions: RNA-RNA recognition by Rop.

The ColE1 plasmid of E. coli encodes a small RNA-binding protein, Rop, which is involved in the regulation of plasmid copy number. Rop, a 4-helix bundle protein, facilitates sense-antisense RNA pairing by binding to the transiently formed hairpin pairs of RNA I and the complementary RNA II. We have identified the residues of Rop that are involved in RNA recognition. The residues form a narrow stripe down one face of the bundle and are symmetrically arranged, with recognition centered about two phenylalanine residues. Our results suggest that these phenylalanine residues interact with the loop region of the hairpin pair, with additional interactions between eight polar residues and the phosphate backbone. By modifying the identity of residue 14, we have created a variant of Rop that displays altered RNA binding specificity. The results of our studies allow us to present a detailed picture of RNA-protein recognition in a novel model system.

Amino Acid Sequence↗
PubMed1986

Mathematical model for the control of ColE1 type plasmid replication.

A mathematical model for the molecular events controlling replication of ColE1 type plasmids is described. All the model parameters can be evaluated independently. The model simulates plasmid replication and accurately predicts the copy-number of ColE1 plasmids carrying a variety of regulatory mutations. The model is used to test the plausibility of hypotheses concerning the interactions of regulatory elements involved in the replication apparatus. The model favorably supports the mechanism proposed by Tomizawa and co-workers concerning the nature of RNA-RNA interactions and that the Rom protein increases the binding between the two RNA species. The hypothesis that the interactions of RNA I-II increases the susceptibility of RNA II to the action of endonucleases is not a plausible mechanism.

DNA Replication↗
PubMed1987

Suppression of ColE1 RNA-RNA mismatch mutations in vivo by the ColE1 Rop protein.

In the bacterial plasmid ColE1 the control of initiation of DNA replication is mediated by the interaction of two complementary RNA molecules, the replication primer and RNA1. The rate of interaction between RNA1 and the primer RNA in vitro can be increased by the product of the ColE1 rop gene, a 63-amino-acid polypeptide. We have investigated the role of the Rop protein in suppressing the incompatibility defects of 13 RNA1-mutant alleles. These RNA1 mutants are defective due to single nucleotide mismatches with their target, the primer RNA. The rop gene suppresses the defective behavior of most of the RNA1 point mismatch mutants in vivo. However, certain mutations that map in stems I and III of RNA1 are not suppressed by rop. The interaction of wild-type and mutant species of RNA1 with ColE1 replication primer transcripts was studied in vitro in the presence or absence of purified Rop protein. The Rop protein is known to increase the rate of wild-type RNA1-primer interaction about twofold in vitro. This enhancement was also observed for mutant RNA1 species having point alterations or a deletion of the 5' terminus of RNA1, which is consistent with the in vivo suppression results. The implications of these results on the mechanism of Rop activity are considered.

Bacterial Proteins↗
PubMed1991

Control of ColE1 plasmid replication by antisense RNA.

One of the two major classes of regulatory strategies that control plasmid copy number involves recognition via base pairing between two plasmid-encoded complementary RNAs. The detailed analysis of this control circuitry has revealed some features of regulatory mechanisms based on RNA-RNA interaction that distinguish them from those based on protein-nucleic acid interaction. These features provide a framework with which to understand other regulatory mechanisms based on RNA-RNA interaction, and will aid in the design of efficient artificial antisense RNA systems.

Bacterial Proteins↗
PubMed1987

A three-dimensional model of domain III of the Escherichia coli small ribosomal subunit.

A three-dimensional model of domain III (nucleotides 920 to 1395) of the 30S ribosomal subunit of E. coli is proposed. The data used as a guide in folding the secondary structure of the RNA into a tertiary structure are four long range RNA-RNA interactions proposed by us on the basis of experiments performed in this laboratory plus two sets of data from other laboratories: protein-RNA cross-linking sites for proteins S1, S3, S7, S10 and S12, and the interprotein distances determined by neutron scattering. The model is consistent with nearly all of the published experimental findings on the structure of domain III.

Base Sequence↗
PubMed1993

Domain V of Giardia lamblia large-subunit rRNA: structure of the peptidyl transferase loop from an early-branching eukaryote and correlation with antibiotic sensitivity.

Large subunit rRNA (LSR) sequences that have been implicated in peptide bond formation form a specific secondary structure called the peptidyl transferase loop (PTL). Although well conserved, the PTLs of eubacteria, archaebacteria, and eukaryotes have several distinct differences. These differences correlate with different sensitivities to peptidyl transferase and translocase inhibitors. To shed light on the basis for these kingdom-specific differences in PTL structure and function, we have analyzed the sequence and secondary structure of LSR domain V, which contains the PTL, from Giardia lamblia. This parasitic protozoan derives from a very early branch in eukaryotic evolution, and its rRNA was previously shown to have bacteria-like features. In vitro and cell-free systems were also used to test the sensitivity of G. lamblia protein synthesis to specific PTL-targeted inhibitors. Our results indicate that the PTL structure and inhibitor sensitivity typical of higher eukaryotes is conserved in G. lamblia. However, several adjacent domain V sequences more closely resemble archaebacterial rRNA, confirming the 'primitive' nature of G. lamblia rRNA. Thus, the eukaryotic PTL has been conserved over a vast evolutionary period. We speculate that the eukaryotic PTL is primordial and employs specific RNA-RNA interactions to catalyze protein synthesis. Three potential interactions were identified.

Animals↗
PubMed1995

Structure of the Leptomonas seymouri trans-spliceosomal U2 snRNA-encoding gene; potential U2-U6 snRNA interactions conform to the cis-splicing counterpart.

We have characterized the U2 small nuclear RNA (snRNA)-encoding gene from the monogenetic trypanosomatid, Leptomonas seymouri (Ls), to begin to identify the RNA-RNA interactions that direct trans-splicing in kinetoplastid protozoa. The U2 gene, which is single copy in this organism, was isolated and sequenced. Although the Ls U2 snRNA contains many of the sequence and secondary structure elements that are conserved among the U2 snRNAs of cis-splicing organisms, it lacks the stem-loop III region and the intron branch point-recognition region, as do other trypanosomatid U2 snRNAs. A transcriptional promoter element within the Trypanosoma brucei U2 gene [Fantoni et al., Mol. Cell. Biol. 14 (1994) 2021-2028] is conserved in the homologous Ls gene. A crucial step in cis-splicing reactions involves specific base-pairing interactions between the U2 and U6 snRNAs. We show here that in trypanosomatids, where no cis-splicing occurs, these same interactions are possible. This highlights key similarities between the two RNA processing events.

Animals↗
PubMed1993

Splicing of pre-mRNA: mechanism, regulation and role in development.

Over the past year, significant progress has been made in the understanding of how RNA-binding factors may facilitate splice-site selection and spliceosome assembly, and confer fidelity to the pre-mRNA splicing reaction. In addition, a number of studies have revealed a complex network of RNA-RNA interactions in the spliceosome, strengthening the structural and functional parallels between nuclear pre-mRNA splicing and the self-splicing group I and group II introns. These new data further support the idea that pre-mRNA splicing occurs by RNA-mediated catalysis and illustrate quite dramatically the dynamic nature of conformational changes in the spliceosome cycle. With respect to tissue-specific pre-mRNA splicing, a number of studies have begun to illuminate mechanisms underlying control of splice-site selection and how so-called 'general' RNA-binding proteins, such as heterogeneous nuclear ribonucleoproteins, may be involved in determining different splicing patterns. Finally, an emerging theme involving the role of splicing in development is that differential transcriptional programs can be triggered in different cell types by alternative splicing patterns that generate transcription factor isoforms with different activities or DNA-binding specificities.

Animals↗
PubMed2004

Determinants of apical loop-internal loop RNA-RNA interactions involving the HCV IRES.

Domain II of the hepatitis C virus internal ribosome entry site is a major RNA structure involved in the viral mRNA translation. It comprises four different structural domains. We performed in vitro selection against the apical loop of the domain II and we identified RNA aptamers folding as an imperfect hairpin with an internal loop of interacting with the apical loop of the domain II. This RNA-RNA interaction creates apical loop-internal loop complex. The aptamer binds the target with an apparent K(d) of 35nM. In this study, the main structural elements of the target and the aptamer involved in the formation of the complex are characterized by mutation, deletion, and RNase probing analysis. We demonstrate that a complementary loop flanked by G,C rich upper and lower stems are crucial for such RNA-RNA interactions.

Base Sequence↗
PubMed2005

Fluorescence resonance energy transfer studies of aminoglycoside binding to a T box antiterminator RNA.

The T box transcription antitermination mechanism is found in many Gram-positive bacteria. The T box genes are typically tRNA synthetase, amino acid biosynthesis, and amino acid transport genes that have a common transcriptional control mechanism in which a unique RNA-RNA interaction occurs between an uncharged tRNA and the 5' leader region of the nascent mRNA, leading to antitermination of transcription. The tRNA binds the mRNA in at least two regions: the specifier sequence and the antiterminator. If the latter interaction does not occur, then transcription is terminated. The binding of eight different aminoglycosides to a model of the Bacillus subtilis tyrS T box antiterminator RNA has been studied using fluorescence resonance energy transfer. The observed single-site binding dissociation constants were in the low to mid micromolar range. The structure-activity relationship of aminoglycoside binding indicates that selective binding of small molecules to T box antiterminator RNA can be achieved.

Aminoglycosides↗
PubMed2004

Site-specific incorporation of a photo-crosslinking component into RNA by T7 transcription mediated by unnatural base pairs.

A photo-sensitive ribonucleotide of 5-iodo-2-oxo(1H) pyridine (Iy) capable of site-specific incorporation into transcripts was developed. The site-specific Iy incorporation into RNA was achieved by T7 transcription mediated by unnatural base pairing between Iy and its partner, 2-amino-6-(2-thienyl)purine (s). By this specific transcription, Iy was incorporated into an anti(Raf-1) RNA aptamer, which binds to human Raf-1 and inhibits the interaction between Raf-1 and Ras. Protein-dependent photo-dimerization of the aptamer was observed when Iy was located at specific positions in the aptamer, showing that the site-specific incorporation of the photo-sensitive component into RNA achieves highly specific crosslinking. This specific transcription mediated by the unnatural base pair would be a powerful tool for generating high-affinity RNA ligands and for analyzing RNA-RNA and RNA-protein interactions, as well as for constructing RNA-based nanostructures.

Bacteriophage T7↗
PubMed2005

In vivo RNA-RNA duplexes from human alpha3 and alpha5 nicotinic receptor subunit mRNAs.

Natural antisense transcripts, because of their potential to form double-stranded RNA (dsRNA) molecules, recently emerged as a mechanism acting on eukaryotic gene regulation at multiple levels. CHRNA3 and CHRNA5, coding for alpha3 and alpha5 subunits of the neuronal nicotinic acetylcholine receptor, have been reported to overlap at their 3'ends in human and bovine genomes. In the present paper, four CHRNA3 and three CHRNA5 human transcripts were characterised, leading to the identification of different antisense complementary regions. Since the two genes are coexpressed in some neuronal and non-neuronal tissues, we ventured on the in vivo identification of RNA-RNA duplexes in both humans and cattle. Using an RNase protection-based approach, CHRNA3/CHRNA5 duplexes were detected in human neuroblastoma SY5Y cells, but not in bovine cerebellum. A semi-quantitative analysis of overlapping transcript levels was performed by real-time RT-PCR. Possible consequences of sense-antisense interaction are discussed.

Animals↗