R17 RNA Research Articles

Subunit I of Qβ Replicase and 30 S Ribosomal Protein Sl of Escherichia coli: EVIDENCE FOR THE IDENTITY OF THE TWO PROTEINS

Abstract Subunit I of the RNA phage-specific Qβ replicase is shown to be identical with the Escherichia coli 30 S ribosomal protein Sl by the following criteria: (a) ability to restore Qβ RNA-directed activity of Qβ replicase lacking subunit I, (b) immunological cross-reactivity, (c) identity of the first four amino acids at the NH2 terminus, and (d) inhibition of R17 RNA- and poly(U)-dependent amino acid incorporation. The mobility of both proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as their amino acid composition is indistinguishable.

Direct physical evidence for secondary structure in an isolated fragment of R17 bacteriophage mRNA.

The region of R17 RNA protected from RNase digestion by coat protein assumes a stable, predictable secondary structure. It seems that this structure is recognised by the protein when functioning as a translational srepressor.

Host interference with viral gene expression: Mode of action of bacterial factor i

The mechanism of interference with R17 viral RNA expression by a host protein, factor i, was studied. Formation of initiation complexes on native bacteriophage R17 RNA molecules, as well as translation of R17 RNA in vitro, is blocked almost quantitatively by factor i. This inhibition is readily overcome by the addition of excess R17 RNA. Extensive complex formation between factor i and R17 RNA occurs during inhibition of initiation complex formation. Moreover, the extent of inhibition of R17 RNA translation correlates closely with the extent of complex formation between factor i and R17 RNA, and exhibits the same sigmoid concentration dependence on factor i.Although initiation complex formation is totally dependent upon initiation factor IF-3, neither this function of IF-3, nor its ability to prevent the association of 30 S and 50 S ribosomal subunits into single ribosomes, is affected by factor i. IF-3, even when present in tenfold molar excess over factor i, fails to relieve the inhibition of initiation on R17 RNA.It is concluded that factor i is a translational represser acting directly on messenger RNA. It is suggested that this repression is cistron-specific, affecting only viral coat protein synthesis. Messenger RNA discrimination by factor i does not involve initiation factor IF-3.

Structural dynamics of bacterial ribosomes: II. Preparation and characterization of ribosomes and subunits active in the translation of natural messenger RNA

Ribosomes from Escherichia coli were tested for activity in initiation with R17 RNA as messenger. All vacant 70 S ribosomes but not all subunits were found to be active. The ability of 30 S and 50 S subunits to form a 70 S couple at Mg 2+ concentrations above 4 m m is a stringent test for activity. Fresh extracts, prepared at 10 m m-Mg 2+ from cells harvested after slow cooling contain up to 80% of the ribosomes in the form of vacant 70 S couples and 20% of free subunits. The proportion of subunits increases with standing as a result of the preferential inactivation of the 50 S particles. “Native” subunits are heterogeneous and consist mostly of active 30 S and inactive 50 S particles. In contrast to 50 S subunits, 30 S subunits prepared by exposure of 70 S ribosomes to low Mg 2+ concentrations, are largely inactive and unable to reassociate with their active 50 S counterparts. However, both initiation and association activity can be restored by heating. The results imply that the structures necessary for subunit association are most critical for the biological activity of ribosomes, presumably because they are topologically closely related to the binding sites for messenger RNA, transfer RNA, and the protein factors for initiation, translocation and termination.

Discriminatory ribosome rebinding of isolated regions of protein synthesis initiation from the ribonucleic acid of bacteriophage R17.

To determine whether bacterial ribosomes recognize a distinguishing feature in the immediate vicinity of actual initiator codons or are directed to these sites through involvement of other portion(s) of the mRNA molecule, the interaction between ribosomes and defined (32)P-labeled initiator fragments from R17 RNA was studied. When incubated with mixtures of the three sites, ribosomes from Bacillus stearothermophilus (which initiate only the A protein on intact phage RNA) are able to select out the A fragment and discriminate against the coat and replicase initiator regions. By contrast, Escherichia coli ribosomes do not rebind that coat-protein region of R17 most efficiently, as they in the native RNA, but likewise prefer the A-protein initiator fragment. In both cases, ribosome binding of the isolated A site is comparable by several criteria to normal polypeptide-chain initiation on an intact R17 messenger RNA in vitro. E. coli ribosomal preference for the A site is confirmed in experiments with randomly fragmented R17 RNA, by both the initiation dipeptide and ribosome protection assay. Thus the A-protein ribosome-binding site of R17 RNA appears intrinsically to be a good initiator, while efficient recognition of the coat and replicase regions requires the participation of some portion of the remainder of the phage RNA molecule.

Raman studies of nucleic acids VIII estimation of RNA secondary structure from raman scattering by phosphate-group vibrations

Analysis of the Raman spectra of a large number of ribonucleotide monomers and polymers indicates that the frequencies and intensities characteristic of the P-O stretching vibrations provide a basis for the quantitative determination of RNA secondary structure. The intensity ratio of the Raman lines at 815 and 1100 cm −1 is shown to have a value of 1.64 ± 0.04 in completely ordered structures and a value of zero in disordered structures. Ordered structures in this context include double-helical configurations containing A · U or G · C pairs and ordered single-stranded configurations (such as occur in poly(rA) and poly(rC)) but exclude other type of base pairing as well as random-chain configurations. The percentages of RNA nucleotides in such ordered configurations, as computed from Raman intensity measurements, are 16-S RNA ( 95 ± 5), 23-S RNA ( 85 ± 5), tRNA fMet ( 84 ± 3), tRNA Val ( 84 ± 3), tRNA Phe ( 85 ± 3) and R17 RNA ( 87 ± 3 %). Since the present method makes use of spectral transitions originating in the RNA backbone and not in the bases, no limitations are imposed by lack of knowledge of the RNA base sequence. In combination with data obtained from quantitative infrared spectroscopy the Raman results are also shown to yield information on the percentages of RNA nucleotides in ordered single-stranded configurations.

Structural dynamics of bacterial ribosomes: I. Characterization of vacant couples and their relation to complexed ribosomes

Ribosomes not engaged in protein synthesis (vacant couples), in contrast to complexed ribosomes bearing nascent chains, dissociate during sedimentation in sucrose gradients at high g forces and at Mg 2+ concentrations below 15 m m. As a result of this dissociation, a new peak between the 70 S complexed ribosomes and the free 50 S subunits is observed, the position of which shifts from about 55 S to 70 S as the Mg 2+ concentration in the gradient is raised from 5 to 15 m m. The apparent 60 S peak consists of 50 S subunits produced during dissociation in the gradient. At low g forces, the sedimentation rate of complexed and vacant ribosomes is indistinguishable, even at 5 m m-Mg 2+. These sedimentation properties are valid criteria to differentiate vacant and complexed ribosomes. This is shown by converting complexed ribosomes quantitatively into vacant couples by removing the nascent chains through termination release or with puromycin, or by converting vacant couples into initiation complexes with R17 RNA, fMet-tRNA and initiation factors. Ribosomes from cells harvested by slow cooling consist almost entirely of vacant couples, all of which are active in protein synthesis with natural messengers. The structural features responsible for the interaction between subunits are discussed.

Translation of bacteriophage R17 and Qβ RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E 3 from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.

Partial inhibition of polysomal ribosomes of Escherichia coli by streptomycin.

With purified initiation-free polysomes of E. coli, whether endogenous or formed in vitro on phage R17 RNA, streptomycin causes partial inhibition of chain elongation. The degree of inhibition is constant over a broad range of streptomycin concentration and decreases markedly with increasing Mg(++) concentration. Hence, streptomycin evidently complexes readily with polysomal ribosomes, causing a partial block in chain elongation. Streptomycin has already been shown to cause a complete block of chain elongation by free ribosomes forming initiation complexes in its presence. The production of a different effect on polysomal ribosomes explains how streptomycin can exert two mutually exclusive effects in cells: phenotypic suppression at low concentrations and irreversible inhibition of protein synthesis at higher concentrations. It also becomes possible to understand why killing by streptomycin is antagonized by agents (such as chloramphenicol) that cause a stable blockade of the ribosomes in polysomes.

Length measurements of RNA synthesized in vitro by Escherichia coli RNA polymerase

A method for visualizing in vitro synthesized RNA in extended form still attached to the DNA template is described. Bacteriophage T4 gene 32 product is attached to the RNA and after fixation with glutaraldehyde the transcription complexes are prepared for electron microscopy by the Kleinschmidt technique. Secondary structure caused by partial complementarity of the RNA can be stretched out by the attachment of bacteriophage T4 gene 32 protein as is shown in the case of bacteriophage R17 RNA. The possibilities of the method are exemplified using T7 as a template for Escherichia coli RNA polymerase. Data for the position of the promoter and the extent of transcription in the region of early T7 messenger RNA are confirmed. In addition, we have demonstrated the presence of a weak promotor at the right-hand end of the DNA; as in the early region, the direction of RNA synthesis is from left to right. Using SV40 viral DNA as a template, up to six RNA chains can be synthesized in the presence of rifampicin. After synthesis for 15 minutes at 37 °C, the length of the RNA chains shows that the polymerase has transcribed at least four times around the DNA circle.

Nucleotide sequences of similar size from the coliphage R17 genome.

A sequence of 33 nucleotides from the coliphage R17 RNA genome was determined. It constitutes the main component of a mixture of fragments that migrate together on electrophoresis in a separation according to molecular weight. Fragments of comparable chain length from 3' end of RNA from coliphage R17, from a region preceding and overlapping the coat-protein cistron ribosome binding site and from the beginning of the A-protein cistron, were also found and characterized. ;Hairpin'-like secondary structures are proposed for the longer fragments, one of which appears to have a tetranucleotide excised in the loop region.

Studies of virus structure by Raman spectroscopy I. R17 virus and R17 RNA

The laser-excited Raman spectrum of the RNA virus, R17, is shown to contain a large number of Raman lines assignable to scattering by vibrations of the nucleotide residues of RNA and the amino-acid residues of protein capsomers. The Raman lines from specific nucleotide vibrations in the phage are compared with their counterparts in the spectrum of protein-free RNA to suggest many similarities of RNA structure in the phage and protein-free states. However, the average configuration of guanine residues in the phage is apparently very different from that of protein-free RNA, suggesting that guanine plays an important role in RNA-protein interactions.

Chain Initiation Factor 2: PURIFICATION AND PROPERTIES OF TWO SPECIES FROM ESCHERICHIA COLI MRE 600

Abstract Chain initiation factor 2 was resolved into two proteins, designated IF-2-α and IF-2-β, by ion exchange chromatography in 6 m urea. The molecular weight of IF-2-α was 98,000 and that of IF-2-β was 83,000. In the presence of IF-1 and IF-3, both IF-2-α and IF-2-β promoted the binding of fMet-tRNA to ribosomes with either ApUpG, GpUpG, or R17 RNA as messenger. The activities of IF-2-α and IF-2-β were additive. Neither protein was protected by GTP against heat inactivation, nor could a stable complex of GTP, IF-2 and fMet-tRNAf be isolated by Sephadex G-50 chromatography. The binding of pure fMet-tRNAf to ribosomes was stimulated by both species of IF-2.

A sequence of 50 nucleotides from coliphage R17 RNA.

A sequence of 50 nucleotides of the RNA from coliphage R17 has been determined. It can be read through in only one translational frame. The sequence is not part of the coat protein cistron nor does it come from the untranslated sequences described so far.Two possible “hairpin” secondary structures have been constructed.The two‐dimensional technique on thin‐layer plates has been used to fractionate intermediates in limited exonucleolytic digests of two dodekamers which could not be sequenced by conventional techniques. The potential general use of this method has been discussed.

Specific recognition of non-initiator regions in RNA bacteriophage messengers by ribosomes of Bacillus stearothermophilus

The interaction between ribosomes of Bacillus stearothermophilus and the RNA genomes of R17 and Qβ bacteriophage has been studied. Whereas Escherichia coli ribosomes can initiate the synthesis of all three RNA phage-specific proteins in vitro, ribosomes of B. stearothermophilus were previously shown to recognize only the A (or maturation) protein initiation site of f2 or R17 RNA. Under these same conditions, a Qβ region is bound and protected from nuclease digestion. Qβ RNA, however, does not direct the synthesis of any formylmethionyl dipeptide in the presence of B. stearothermophilus ribosomes, nor does the binding of either this Qβ region or the R17 A protein initiation site to these ribosomes show the same fMet-tRNA requirement for recognition of initiator regions as that previously established with E. coli ribosomes. Analysis of a 38-nucleotide sequence in the protected Qβ region reveals no AUG or GUG initiator codon. These observations suggest that messenger RNA may be recognized and bound by B. stearothermophilus ribosomes quite independently of polypeptide chain initiation. Binding experiments using R17 RNA and mixtures of components from B. stearothermophilus and E. coli ribosomes confirm the conclusion drawn by Lodish (1970 a) that specificity in the selection of authentic phage initiator regions by the two species resides in the ribosomal subunit(s). However, anomalous attachment of B. stearothermophilus ribosomes to R17 RNA, which is observed upon lowering the incubation temperature of the binding reaction, is clearly a property of the initiation factor fraction. The results are discussed with respect to current ideas on the role of ribosomes and initiation factors in determining the specificity of polypeptide chain initiation.

Nucleotide sequences of fragments of R17 bacteriophage RNA from the region immediately preceding the coat-protein cistron.

The known nucleotide sequence preceding the coat protein cistron in R17 bacteriophage RNA has been extended by 23 residues towards the 5′‐terminus of the RNA. 32P‐labelled R17 RNA was partially digested with Escherichia coli ribonuclease IV and the resulting fragments were resolved by electrophoresis on polyacrylamide gels. Fingerprinting techniques were then used to identify fragments with sequences overlapping the known sequence for the ribosomal‐binding site at the beginning of the coat protein cistron. Three such fragments (59, 31 and 26 nucleotides long) were found routinely and a slightly larger fragment was found in one experiment using a different batch of enzyme. The nucleotide sequences of the fragments were studied by established radiochemical techniques and all four fragments were found to have the same 3′‐terminus. The nucleotide sequence determined for the largest fragment is A‐A‐U‐C‐A‐G‐G‐C‐A‐A‐C‐G‐G‐C‐U‐C‐U‐C‐U‐A‐A‐A‐U‐A‐G‐A‐G‐C‐C‐C‐U‐C‐A‐A‐C‐C‐G‐G‐A‐G‐U‐U‐U‐G‐A‐A‐G‐C‐A‐U‐G‐G‐C‐U‐U‐C‐U‐A‐A‐C‐U‐U‐U where AUG is the initiation codon for the coat protein cistron. The sequence preceding this AUG contains three possible nonsense codon but whether any of them is actually used to terminate synthesis of the R17‐A protein is not known; most likely the untranslated sequence between the A protein and coat protein cistrons is at least 26 residues long. The sequence reported here shows little homology with the known sequences preceding the R17‐A protein and RNA synthetase cistrons nor with any of the sequences preceding the cistrons in Qβ phage RNA; however, it is almost identical to the sequence preceding the coat protein cistron of the closely related phage f2. The nucleotide sequence which we have determined can be folded into the form of two hydrogen‐bonded loops.

Studies on the bacteriophage MS2. Some nucleotide sequences from the RNA-polymerase gene

The knowledge of the amino acid sequence from the coat protein of the RNA bacteriophages f2, R 17 and MS2 [ l-31 allowed the identification of several RNA fragments as being derived from this gene. They were all obtained by partial ribonuclease hydrolysis of R17 RNA [4-61, f2 RNA [7] or MS2 RNA [8, 91. For MS2, these sequences could be further extended, and the complete sequence of the coat gene was established [lo]. Furthermore, this se- quence was followed by a 36 nucleotides long inter- cistronic region and by the ribosomal binding site of the next gene, the RNA-polymerase. As the amino acid sequences for the two other gene prod- ucts, the A-protein and the viral RNA-polymerase, are not known, no such simple test exists to identify polynucleotides corresponding to the latter two genes. In this communication we present data on three nucleotide fragments derived from the polymerase gene. This conclusion is based on direct overlaps with known sequences [ lo] , on the established gene order for these phages [ 11, 121, and on the mapping of a series of polypurine tracts in two fragments of the RNA molecule [ 131 . 2. Methods All RNA fragments described in this study were *

Mechanism and control of initiation in the translation of R17 RNA.

A general model of initiation of protein synthesis based on experiments with R17 RNA as messenger proposes a two-stage mechanism, the first stage requiring dissociation into subunits and initiation factors, the second involving direct attachment of ribosomes.

Nucleotide sequences of two fragments from the coat-protein cistron of bacteriophage R17 ribonucleic acid

Bacteriophage R17 RNA was labelled with (32)P and was subjected to partial digestion with ribonuclease T(1). The products were fractionated by ionophoresis on polyacrylamide gel. Two fragments were purified and their nucleotide sequences determined by methods involving complete and further partial digestion with ribonucleases A and T(1). Fragment 20 had a sequence that coded for the amino acids in positions 32-53 of the coat protein of the bacteriophage. Fragment 20X, on further purification in 7m-urea, gave rise to two smaller nucleotides whose sequences coded for the amino acids in positions 56-66 and 67-76 of the coat protein. The sequence of the two fragments was such that they could be written in the form of loops stabilized by base-pairing.

Extent of variation in three related bacteriophage RNA molecules

The isolation and sequence analysis of a series of oligonucleotides from complete ribonuclease T 1 digests of bacteriophage R17 RNA have been reported (Jeppesen, 1971). We have obtained the homologous sets of large T 1-resistant oligonucleotides from the related phages f2 and MS2. As expected from earlier findings (Nichols & Robertson, 1971), each RNA can be uniquely characterized by its ribonuclease T 1 fingerprint. Comparison of nucleotide sequences from the sets of RNA fragments from R17, f2 and MS2 allows an estimate of the over-all degree of variation among the three RNA's (2.8 to 3.4%), and yields additional evidence that non-coding regions show significantly lower variation. R17, f2 and MS2 are apparently not linear descendants, but could have diverged from a common ancestor. Base substitutions involving U and C transitions occur at a higher frequency than would be expected on a random basis. It appears probable that a total of 15 to 20 base replacements in the phage coat protein cistron may accompany the single amino-acid difference between f2 coat protein and that of either R17 or MS2. We conclude that analysis of this sort should prove useful in determining the extent of variation among members of any set of closely related nucleic acid molecules.