Hugh D. Robertson

Cell-Free Circularization of Viroid Progeny RNA by an RNA Ligase from Wheat Germ

Linear, potato spindle tuber viroid RNA has been used as a substrate for an RNA ligase purified from wheat germ. Linear viroid molecules are efficiently converted to circular molecules (circles) which are indistinguishable by electrophoretic mobility and two-dimensional oligonucleotide pattern from viroid circles extracted from infected plants. In light of recent evidence for multimeric viroid replication intermediates, cleavage followed by RNA ligation by a cellular enzyme may (i) be a normal step in the viroid life cycle and (ii) may also reflect cellular events.

Sequence and symmetry in ribosome binding sites of bacteriophage f1 RNA

Abstract RNA was synthesized in vitro from α-32P-labeled ribonucleoside triphosphates with Escherichia coli RNA polymerase from covalently closed, circular, double-stranded DNA isolated from cells infected with bacteriophage f1. This RNA, which serves as an efficient message in vitro, was bound to ribosomes and the initiation complexes were digested with bovine pancreatic ribonuclease. Ribosome-protected fragments were isolated, purified and separated by two-dimensional analysis using electrophoresis and homochromatography. Sequence analysis, taking advantage of the ability to determine nearest neighbors, was done by conventional techniques. The sequences of the ribosome-protected fragments were found to fall into three classes. One sequence corresponds to the amino-terminal region of the protein product of f1 gene V, a DNA binding protein. It is proposed that a second sequence may correspond to the amino-terminal region of a precursor to the major coat protein. No assignment has yet been made for the third sequence. Comparisons are made between these three sequences and others that are available, both in terms of sequence features that have been pointed out earlier, and in terms of certain considerations of symmetry and syntax † prominent in these binding site sequences that have not been discussed before.

Determination of nucleotide sequences from double-stranded regions of HeLa cell nuclear RNA

Double-stranded regions which comprise about 4% of isolated HeLa cell heterogeneous nuclear RNA have been characterized by RNA fingerprinting and sequencing analysis. The simplicity of the pattern in two-dimensional RNA fingerprints suggests a sequence complexity of about 1000 nucleotides. The nucleotide sequences of six prominent RNase T1-resistant oligonucleotides (ranging in size from 7 to 9 bases) have been determined using isolated double-stranded nuclear RNA labeled in vivo with 32P-labeled inorganic phosphate. We conclude that (here exists a substantial subpopulation of simple, potentially complementary sequences common to much of the heterogeneous nuclear RNA population and interspersed with other kinds of sequences.

RNA precursor molecules and ribonucleases in E. coli

SummaryThe post-transcriptional alteration of oneE. coli RNA species (tyrosine tRNA) by nucleases is now understood in some detail. Knowledge of this pathway has allowed us to speculate on the nature and control of the processing of otherE. coli RNA species by the combined attack of endo- and exo-nucleolytic ribonucleases. A review of the properties of knownE. coli ribonucleases has led us consider which of these activities might be capable of functioning together in different pathways. The control of these nuclease activities (singly or in groups) may be exerted through structural transitions of RNA substrates or through ionic or protein cofactors.

The binding of 125I-labelled rabbit globin messenger RNA to reticulocyte ribosomes.

Abstract Rabbit globin messenger RNA was labelled in vitro with 125 I to specific activities in the range 20 to 200 × 10 6 cts/min per μg. This 125 I-labelled mRNA bound to rabbit reticulocyte ribosomes with the kinetics and sensitivity to inhibitors expected from its participation in the normal process of the initiation of protein synthesis. Furthermore, when modified in 25% of its cytidine residues with unlabelled iodide, the mRNA coded for the same series of initiation peptides as did the unmodified mRNA. Using the techniques of RNA fingerprinting, the binding reaction was shown to select against contaminants and against “globin mRNA” molecules which lack a particular oligonucleotide implicated in the initiation process. When the 125 I-labelled mRNA was bound to ribosomes, both the initiating 40 S subunits and the 80 S ribosomes protected a fraction of the mRNA from digestion by pancreatic ribonuclease. Fingerprint analysis showed that highly specific regions of the mRNA were protected by the 40 S subunits and 80 S ribosomes and that these two protected regions were not identical.

Escherichia coli ribonuclease III

Publisher Summary This chapter considers methods for the assay of RNase III, preparation of the substrates required, and a purification scheme for the enzyme that has been tested on strains of E. coli carrying cloned copies of the gene for RNase III (the rnc gene), which has recently been identified and sequenced. RNase III processing of various RNA precursors is carried out by polyacrylamide gel electrophoresis followed by characterization of the cleavage products. Centrifugation at 10,000 rpm in the SS34 rotor of the Sorvall RC-5B centrifuge was followed by isolation of the supernatant and rehomogenization of the pellets in 20 ml of additional extraction buffer. As expected, the RNase T1-resistant oligonucleotide containing the RNase III cleavage site in the intact R1.1 RNA is missing and is replaced by terminal fragments with the expected properties in the two cleavage products. With the availability of increased amounts of RNase III enzyme through genetic engineering techniques, it should be possible to use the enzyme with increased efficiency in studies of its own in vivo processing reactions as well as in investigations in which RNase III is used as a reagent to detect double-stranded RNA and to introduce specific cleavages into RNA molecules.

Analysis of ultraviolet-induced RNA-RNA cross-links: a means for probing RNA structure-function relationships

Publisher Summary This chapter presents analysis of ultraviolet-induced RNA–RNA cross-links. To obtain maximum information about local bonding patterns, the UV-induced cross-link must be mapped to the nucleotide level. Direct RNA analysis is needed. However, if cross-links are to be used only to determine which portions of the RNA are close together in the three-dimensional structure, indirect techniques, such as primer extension, may be employed. The mapping of an extensive series of RNA–RNA cross-links introduced into rRNA has led to a detailed three-dimensional picture of 16 S rRNA. While additional procedures are usually required to locate a UV-induced bond in a large RNA molecule, a cross-link in an RNA smaller than 1000 nt can often be detected directly by a change the cross-link makes in the characteristic pattern of ribonuclease-resistant oligonucleotides. Primer extension is useful for mapping cross-links, especially when employed in combination with direct RNA analysis.

RNA-processing Nucleases

In all living organisms there are biosynthetic pathways by which gene transcripts are converted step-by-step into their final functional form in cells. The several classes of functional RNAs, like mRNA, tRNA, or rRNA, each undergo processing events during their intracellular metabolism (Perry 1982). The evidence is overwhelming that for each type of RNA there are ribonucleases that systematically alter the size of the original gene transcript. In addition to size changes, other modifications also occur in RNA biosynthetic pathways (e.g., methylation, polyadenylation, CCA addition, and the acquisition of cap structures at 5′ termini). This article is concerned solely with the nature and mode of action of enzymes that alter the size of primary RNA transcripts and their processing intermediates. Since every gene transcript contains RNA, the main distinguishing feature among transcripts is nucleotide sequence. It is probably sequence, therefore, that determines which biosynthetic pathway an RNA molecule enters—that for rRNA, mRNA, tRNA, or some other RNA species. It must also be sequence that ultimately determines which set of processing RNases acts on a particular gene transcript, although it may not be sequence alone that directly defines a cleavage site. RNA-PROCESSING EVENTS The involvement of RNases in RNA-processing events can be categorized as follows: (1) specific endonucleolytic cleavage of gene transcripts to alter their size; (2) specific exonucleolytic cleavage to perform small size reductions at either end of a molecule; or (3) specific rejoining of RNA molecules (ligation) associated with splicing of transcripts. The first two nucleolytic mechanisms are known...

[12] RNA fingerprinting

Publisher Summary RNA fingerprinting analysis is the fastest way to obtain accurate information about an RNA processing reaction. The presence of a new spot, pGp, in one of the products identifies the exact phosphodiester bond cleaved during the processing reaction and indicates the final deposition of the phosphate group. The original RNA fingerprints were made using high-voltage electrophoresis on diethylaminoethyl (DEAE) paper for the second dimension. This fractionation procedure provides considerable information about the base composition of the oligonucleotides. The protocol chosen for ribonuclease digestion depends on whether the RNA is already radiolabeled. In all cases, RNAs are dried down in small silicon-treated glass tubes. Glass tubes are used because their clarity permits even minute quantities of RNA to be seen. The phosphatase treatment removes the 3′-phosphate groups produced by ribonuclease digestion. Samples are then sealed into drawn-out capillary tubes and are heated in a bath of boiling water for three minutes to inactivate the phosphatase.

Interference between coinoculated viroids

Experiments were carried out to seek evidence of an interaction between two viroid RNAs introduced to tomato plants in the same inoculum. At the level of symptom expression, the severe isolate of potato spindle tuber viroid (PSTV) dominated the mild isolate. Seventy-five percent of the plants inoculated with a 100-fold excess of the mild isolate developed unattenuated symptoms of severe disease. Other experiments revealed that infectious RNA molecules transcribed from cloned DNA templates containing PSTV sequences reduced the level of hop stunt viroid (HSV) RNA present in nucleic acid extracts of plants which had been inoculated with a mixture of dimeric plus-strand transcripts of these two viroids. Plants inoculated with dual transcripts--containing two copies of PSTV linked to two copies of HSV--developed characteristic symptoms of severe PSTV. Dot hybridization demonstrated that only PSTV replicated to detectable levels in these plants. A likely interpretation of these results is that the HSV portion of the dual transcripts failed to replicate because of interference from PSTV. These results raise questions about how the process of viroid replication is related to symptom expression, and lead to suggested models for the effect of viroid-like RNAs in cells under both normal and pathogenic circumstances.