Codon usage and secondary structure of MS2 phage RNA.

MS2 is an RNA bacteriophage (3569 bases). The secondary structure of the RNA has been determined, and is known to play an important role in regulating translation. Paired regions of the genome have a higher G+C content than unpaired regions. It has been suggested that this reflects selection for high G+C content to encourage pairing, but a re-analysis of the data together with computer simulation suggest that it is an automatic consequence in any RNA sequence of the way it folds up to minimise its free energy. It has also been suggested that the three registers in which pairing can occur in a coding region are used differentially to optimise the use of the redundancy of the genetic code, but re-analysis of the data shows only weak statistical support for this hypothesis.     

The regulatory region of MS2 phage RNA replicase cistron. Functional activity of individual MS2 RNA fragments

The present work deals with the structural-functional organization of regulatory regions of messenger RNAs. Some principles of the action of a translational repressor (coat protein) and the formation of the ribosomal initiation complex at the replicase cistron have been studied with MS2 phage RNA. When the complex of MS2 RNA with the coat protein is treated with T₁ ribonuclease, the coat protein selectively protects mainly two fragments (59 and 103 nucleotides in length) from digestion; these fragments contain the intercistronic regulatory region and the beginning of the MS2 replicase cistron. These polynucleotides have been isolated in a pure state and their primary structure has been established.It has been established that both MS2 RNA fragments contain all the necessary information for specific interaction with MS2 coat protein and form a complex with it with an efficiency close to that observed in the case of native MS2 RNA. They also provide the normal polypeptide chain initiation at the replicase cistron. Enzymatic binding of the second aminoacyl-tRNA and electrophoretic analysis of N-terminal dipeptides prove that only the true initiator codon of the replicase cistron is recognized by a ribosome despite the presence of a few additional AUG triplets within the polynucleotides. Under conditions of limited hydrolysis by T₁ ribonuclease, the beginning of the replicase cistron has been removed from the shortest polynucleotide leading to a complete loss of its ability to bind both the coat protein and a ribosome.Some principles of the functioning of the regulatory region in MS2 RNA as well as the nature of the initiator signal of protein biosynthesis are discussed.     

Overlapping genes in RNA phage: a new protein implicated in lysis.

We have identified a new 75 amino acid polypeptide (L protein) following f2 phage infection of E. coli. It is encoded by an out-of-phase overlapping gene which begins within the coat protein gene, ends in the replicase gene and covers the 36 base intercistronic space between them. A mutant f2 phage carrying a UGA mutation (op3), which complements mutations in the other three f2 genes (coat, A protein and replicase), fails to lyse cells (Model, Webster and Zinder, 1979) and also fails to produce L protein. Both lysis and L protein are restored following op3 infection of a UGA suppressor-containing strain or infection of wild-type bacteria with a revertant of op3. L protein is found in the insoluble fraction of artificially lysed cells. In this paper, we present the time course of its synthesis relative to the other f2-coded polypeptides: L protein synthesis increases as replicase synthesis decreases. We also report the discovery of another phage-coded polypeptide, which appears to be the product of a novel mode of translation: initiation at the coat protein initiation site, followed by translational frame shifting into the L protein frame and termination at the L protein terminus.     

Studies of virus structure by laser-Raman spectroscopy. II. MS2 phage, MS2 capsids and MS2 RNA in aqueous solutions.

Laser-Raman spectra of the bacteriophage MS2, and of its isolated coat-protein and RNA components, have been obtained as a function of temperature in both H₂O and D₂O (deuterium oxide) solutions. The prominent Raman lines in the spectra are assigned to the amino acid residues and polypeptide backbone of the viral coat protein and to the nucleotide residues and ribosyl-phosphate backbone of the viral RNA. The Raman frequencies and intensities, and their temperature dependence, indicate the following features of MS2 structure and stability. Coat-protein molecules in the native phage maintain a conformation determined largely by regions of β-sheet (~60%) and random-chain (~40%) structures. There are no disulfide bridges in the virion and all sulfhydryl groups are accessible to solvent molecules. Protein-protein interactions in the virion are stable up to 50 °C. Release of viral RNA from the virion does not affect either the conformation of the coat-protein molecules or the thermal stability of the capsid. MS2 RNA within the virion contains a highly ordered secondary structure in which most (~85%) of the bases are either paired or stacked or both paired and stacked and in which the RNA backbone assumes a geometry of the A-type. When RNA is partially or fully released from the virion its overall secondary structure at 32 °C is unchanged. However, the exposed RNA is more susceptible to changes in secondary structure promoted by increasing the temperature. Thus the viral capsid exerts a significant stabilizing effect on the secondary structure of MS2 RNA. This stabilization is ionic-strength dependent, being more pronounced in solutions containing high concentrations of KCl. Raman intensity profiles as a function of temperature reveal that disordering of the MS2 RNA backbone and rupture of hydrogen-bonding between complementary bases are gradual processes, the major portions of which occur above 40 °C. However, the unstacking of purine and pyrimidine bases is a more co-operative phenomenon occurring almost exclusively above 55 °C.     

Secondary structure of MS2 phage RNA and bias in code word usage.

Based on the secondary structural model of MS2 RNA, it is shown that, in base-pairing regions of the RNA, there is a bias in the use of synonymous codons which favours C and/or G over U and/or A in the third codon positions, and that in non-pairing regions, there is an opposite bias which favours U and/or A over C and/or G. This nature is interpreted as a result of selective constraint which stabilises the secondary structure of the single-stranded RNA genome of the MS2 phage.     

The accessibility of phage MS2 RNA to structure specific nucleases in various conditions

The accessibility of ds-and ss-segments of phage MS2 RNA to ds-and ss-specific nucleases (RNase III, nuclease SV and nuclease S1) was studied. The results show that the RNA has hydrolysis sites for all the nucleases used. These sites are unvariable in a wide range of the conditions (ionic strength, pH, bivalent cations and temperature) and are not changed also after denaturation-renaturation of the RNA. This testifies that the distribution and interactions of ds-and ss-segments in the whole molecule are very specific and stable.     

Control of start codon choice on a plant viral RNA encoding overlapping genes.

The signals that control initiation of translation in plants are not well understood. To dissect some of these signals, we used a plant viral mRNA on which protein synthesis initiates at two out-of-frame start codons. On the large subgenomic RNA (sgRNA1) of barley yellow dwarf virus-PAV serotype, the coat protein (CP) and overlapping 17K open reading frames (ORFs) are translated beginning at the first and second AUG codons, respectively. The roles of bases at positions -3 and +4 relative to the AUG codons in efficiency of translation initiation were investigated by translation of sgRNA1 mutants in a cell-free extract and by expression of a reporter gene from mutant sgRNA1 leaders in protoplasts. The effects of mutations that disrupted and restored secondary structure encompassing the CP AUG independently of, and in combination with, changes to bases -3 and +4 were also examined. Partial digestion of the 5' end of the sgRNA1 leader with structure-sensitive nucleases gave products that were consistent with the predicted secondary structure. Secondary structure had an overall inhibitory effect on translation of both ORFs. In general, the "Kozak rules" of start codon preference predominate in determining start codon choice. Unexpectedly, for a given CP AUG sequence context, changes that decreased initiation at the downstream 17K AUG also reduced initiation at the CP AUG. To explain this observation, we propose a new model in which pausing of the ribosome at the second AUG allows increased initiation at the first AUG. This detailed analysis of the roles of primary and secondary structure in controlling translation initiation should be of value for understanding expression of any plant gene and in the design of artificial constructs.     

Binding of oxytetracycline to E. coli ribosomes

Binding isotherms of oxytetracycline to E. coli MRE-600 ribosomes were measured by equilibrium dialysis. The results are consistent with the presence of two classes of binding sites for the antibiotic on ribosomes having different reactivities. There is one strong binding site for the antibiotic on the ribosome, while the number of weak binding sites is about 500. The association constant for strong complexes is about 10³ times greater than the corresponding value for weak complexes. The strong binding of the antibiotic prevents the template dependent association of aminoacyl-tRNA with ribosomes.All-Union Institute of Antibiotics Research, Moscow 113105.