Individual RNA Molecules Research Articles

Structure detection through automated covariance search

This paper summarizes our investigations into the computational detection of secondary and tertiary structure of ribosomal RNA. We have developed a new automated procedure that not only identifies potential secondary and tertiary structural interactions, but also provides the covariation evidence that supports the proposed bondings, and any counterevidence that can be detected in the known sequences. A small number of previously unknown higher-order structural features have been detected in individual RNA molecules (16S rRNA and 7S RNA) through the use of our automated procedure. We are systematically studying mitochondrial rRNA, seeking tertiary structure within 16S rRNA and quaternary structure between 16S and 23S rRNA. To test hypotheses suggested by an examination of our program's output, our colleagues in biology are sequencing key portions of the 23S ribosomal RNA for species in which the known 16S ribosomal RNA exhibits variation (from the dominant pattern) at the site of a proposed bonding. Our ultimate hope is that automated covariation analysis will contribute significantly to a refined picture of ribosomal structure.

Conformation of Free Ribosomal RNAs by STEM and Wet Film Technique as a Phylogenetic Probe

The similarity of ribosomes involvement in protein synthesis in evolutionarily distant species makes this cellular organelle an attractive phylogenetic probe. The evolutionary changes in ribosomes are reflected not only in the morphology of the ribosome but also in the composition and structure of its components - proteins and RNAs.We have investigated the extent of structural similarity of free rRNAs from baby hamster kidney (BHK) cells and Escherichia coli as examples of RNAs from a eukaryote and a prokaryote, respectively. Using dedicated STEM and “wet film” technique (Wall et al. these Proceedings) for specimen deposition we have improved considerably the resolution of RNA conformation in comparison to the glow discharge technique. A gallery of individual unstained freeze-dried 28S RNA molecules Isolated from the large (60S) ribosomal subunits from BHK cells, obtained by deposition from distilled water, is presented in Fig. 1a.

Topographical mapping of ribosomal RNAs in situ by electron spectroscopic imaging

Visualization of the in situ location of the individual components of any macromolecular system is important for understanding its assembly, interactions, and function. Ribosomes, which are small cellular organelles involved in protein synthesis are high molecular weight nucleoprotein complexes composed only of proteins and RNAs. This “simple” composition of ribosomes enables us topographical studies directed either towards localization of the individual ribosomal protein and RNA molecules or merely to the determination of the distribution of the protein and RNA moieties within the ribosome and its subunits. We have utilized the recent progress in the development of microanalytical electron spectroscopic techniques, electron energy loss spectroscopy (EELS) in particular, and the unique distribution of the phosphorus atoms on the ribosome (the phosphorus atoms are present only in the structural backbone of the rRNA) for the direct tracing of the RNA molecules in situ.

Localization of small nuclear polymerase I RNA sequences at the 5' end of the human rDNA transcription unit.

A human ribosomal DNA clone isolated from a genomic library was used to localize the DNA sequences coding for HeLa cell small nuclear polymerase I RNA (snPI RNA). By using a subcloned 1.2-kb EcoRI-SalI fragment, including the initiation region of the 45S rRNA transcription unit, it was shown that the snPI RNA sequences are located within the first 600 nucleotides of the 5' end of the external transcribed spacer. Strand-specific hybridization following exonuclease III digestion of the plasmid containing the 1.2-kb Eco-Sal fragment demonstrated that the snPI RNA molecules and the 45S pre-rRNA are transcribed from the same coding strand. A detailed mapping of individual snPI RNA molecules showed that most of these small RNA species span the putative early processing site at position 415 of the external transcribed spacer of the human rRNA precursor.

The Concept of a Blood-Brain Barrier

<h3>ABSTRACT</h3> While RNA secondary structure prediction from sequence data has made remarkable progress, there is a need for improved strategies for annotating the features of RNA secondary structures. Here we present bpRNA, a novel annotation tool capable of parsing RNA structures, including complex pseudoknot-containing RNAs, to yield an objective, precise, compact, unambiguous, easily-interpretable description of all loops, stems, and pseudoknots, along with the positions, sequence, and flanking base pairs of each such structural feature. We also introduce several new informative representations of RNA structure types to improve structure visualization and interpretation. We have further used bpRNA to generate a web-accessible meta-database, “bpRNA-1m”, of over 100,000 single-molecule, known secondary structures; this is both more fully and accurately annotated and over 20-times larger than existing databases. We use a subset of the database with highly similar (≥90% identical) sequences filtered out to report on statistical trends in sequence, flanking base pairs, and length. Both the bpRNA method and the bpRNA-1m database will be valuable resources both for specific analysis of individual RNA molecules and large-scale analyses such as are useful for updating RNA energy parameters for computational thermodynamic predictions, improving machine learning models for structure prediction, and for benchmarking structure-prediction algorithms.

An analysis by electron microscopy of early simian virus 40 RNA from a tsA mutant

A new electron microscopic technique, developed by J. Ferguson and R. W. Davis (manuscript in preparation), has been used to investigate the pattern of early transcription in cells infected with simian virus 40 (SV40). DNA binding protein from E. coli and antibody to this protein increase the apparent thickness of single-stranded DNA. Thus the positions of termini and the sizes of individual molecules of RNA can be determined after hybridization to a defined single strand of DNA. Since the concentration of early RNA is very low in cells infected by wild-type SV40, the RNA was prepared from cells infected with the temperature-sensitive mutant tsA58, where early RNA is greatly over-produced after a shift from permissive to restrictive temperature late in infection. Nuclear or cytoplasmic RNA was hybridized to full-length linear early strands of SV40 DNA. Using the known end points generated by the restriction endonuclease (Eco RI) and an internal marker to define orientation of the strand, the positions of the 5′ and 3′ termini of individual RNA molecules were determined. Most molecules of cytoplasmic early RNA have a 5′ terminus at 0.67 SV40 fractional length, and the majority of the 3′ termini extend well into the late region, at least as far as the Eco RI restriction site at map position 0. The mean 5′ terminus of nuclear early RNA molecules was at 0.68 SV40 fractional length. Many of these molecules are small (0.01–0.20 fractional length), but some extend to the Eco RI restriction site or possibly beyond, as found for the cytoplasmic early RNA. The effects on the composition and size of early RNA pools of the tsA mutation or of exposure to high temperature are unknown.

Mitochondrial polyadenylic acid-containing RNA: Localization and characterization

The RNA from the mitochondrial fraction of animal cells contains a polyadenylic acid sequence, approximately 55 nucleotides in length, which migrates at about 4 S in gel electrophoresis and which is attached to high molecular weight RNA. The experiments reported here indicate that: (a) the 4 S poly(A) sequence is found only in the mitochondrial fraction; (b) the RNA containing 4 S poly(A) is located within structures (presumably mitochondria) which protect it from pancreatic ribonuclease; (c) no RNA containing the longer poly(A) of nuclear origin appears to be located in mitochondria; (d) the 4 S poly(A), but not the longer poly(A), is attached to RNA which hybridizes to mitochondrial DNA; and (e) this poly(A) sequence is located at the 3′ end of the RNA molecule. The poly(A)-containing RNA can be isolated by affinity to oligodeoxyribothymidylic acid cellulose and resolved into approximately eight distinct species by acrylamide gel electrophoresis. These may correspond to individual mitochondrial messenger RNA molecules.

Induction of mutations in an RNA tumour virus by an analogue of a DNA precursor.

IF the replication of RNA tumour viruses involves a DNA implicative intermediate1–3, precursor analogues which modify DNA could inactivate the viral progeny by causing occasional errors in base pairing during transcription of RNA from DNA. Each RNA molecule transcribed might be defective in certain genes, but because different molecules would carry different defects, there would be intracellular complementarity. Nonetheless, incorporation of individual RNA molecules into virions would give defective particles. Such defective particles have been found after infection of cells by Rous sarcoma virus (RSV) and exposure of these cells to 5-bromodeoxy-uridine (BrdU)4, an analogue of deoxythymidine. An extension of these studies is described here. The RSV stock consisted of transforming Bryan RSV0, the non-transforming Rous associated virus (RAV) and phenotypic mixtures.

Some General Principles

Andrew Travers MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England In this article I consider how the interactions of RNA polymerase and promoter sites may determine and control the specificity of the initiation of transcrip- tion in bacteria. I shall concentrate on two aspects of this topic: first how regulation is mediated during normal vegetative growth, as exemplified in particu- lar by ribosomal RNA synthesis; and second how initiation specificity may be altered during the un- folding of a developmental program such as sporu- lation. The first step in the synthesis of a specific RNA molecule is the binding of RNA polymerase holoen- zyme within a specific region of the DNA template. This region is termed a “promoter” site and is clas- sically defined by cis acting mutations that affect the rate of initiation of a single RNA species (Scaife and Beckwith, 1966). In the subsequent discussion I shall use the term promoter to describe the whole DNA region controlling the initiation of an individual RNA molecule. This definition thus includes binding sites both for RNA polymerase and for specific DNA binding regulatory proteins. Molecular Mechanism of Initiation The sequence of molecular events necessary for the accurate initiation of RNA synthesis in vitro (Fig- ure 1) was first formulated by Zillig and his collabo- rators (Fuchs et al., 1967) and subsequently con- firmed and elaborated by Bautz, Bautz, and Beck (1972) and by Hinkle and Chamberlin (1972b). After unproductive random interactions with the DNA template, the RNA polymerase holoenzyme, con- sisting of the core polymerase, c&3’, complexed with the u polypeptide (Burgess, 1969) “recog- nizes” a specific structure or sequence within a promoter region. This recognition, which probably requires the presence of the sigma factor, enables the polymerase to bind to the DNA in this region at least an order of magnitude more tightly than in nonspecific interactions. The structural features re- quired for polymerase recognition of the double- stranded DNA have yet to be characterized, but it is perhaps significant that a class of promoters for E. coli holoenzyme contain either of two nucleotide sequences recognized by Hind endonucleases, GTCGAC or GTTAC (Allet et al., 1974). The next, and crucial, step in the initiation process is a change in the conformational state of the DNA in the promoter region (Travers, Baillie, and Pedersen, 1973). Once this transition has occurred, the poly- merase is then poised to start the synthesis of an