Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages.

The complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Q(beta), which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual position, overlapping the coat and replicase proteins. Instead, two small ORFs are present at the 5' terminus, preceding the maturation gene. One of these might encode a lysis protein. The other is of unknown function. Other new features concern the 3'-terminal sequence. In all ssRNA coliphages, there are always three cytosine residues at the 3' end, but in AP205, there is only a single terminal cytosine. Distantly related viruses, like AP205 and the coliphages, do not have significant sequence identity; yet, important secondary structural features of the RNA are conserved. This is shown here for the 3' UTR and the replicase-operator hairpin. Interestingly, although AP205 has the genetic map of a levivirus, its 3' UTR has the length and RNA secondary structure of an allolevivirus. Sharing features with both MS2 and Q(beta) suggests that, in an evolutionary sense, AP205 should be placed between Q(beta) and MS2. A phylogenetic tree for the ssRNA phages is presented.

Complete nucleotide sequence of the group I RNA bacteriophage fr.

We report the complete nucleotide sequence of the group I RNA bacteriophage fr. The entire genome consists of 3575 nucleotides, six nucleotides more than the only other sequenced group I representative, MS2. The greatest divergence between these phages occurs in the 5' terminal region of the A gene, while the lysis-replicase gene overlap, the coat gene and the central region of the replicase gene are highly conserved. Overall sequence homology between fr and MS2 is 77%. Here, we present a general comparison between the two phages. In the accompanying paper we use phylogenetic sequence comparison between MS2 and fr to deduce the secondary structure at the 3' untranslated region.

Determination of the RNA secondary structure that regulates lysis gene expression in bacteriophage MS2.

The lysis gene of the RNA bacteriophage MS2 is not expressed unless translation of the overlapping coat gene takes place. To understand the molecular basis for this translational coupling the RNA secondary structure around the lysis gene start was analyzed with structure-specific enzymes and chemicals. The existence of a hairpin between nucleotides 1636 and 1707 is in agreement with the structural mapping data and also with the conservation of base-pairing in the related M12 phage. In this hairpin, the G residues in the Shine and Dalgarno region and start codon are inaccessible to RNase T1, which is consistent with the fact that ribosomal access to the lysis gene is blocked when there is no coat gene translation. Deletions or point mutations that are predicted to destabilize the hairpin give rise to lysis protein synthesis that is independent of coat gene translation. Base substitutions that are not expected to weaken the helix do not lead to independent lysis gene expression. Finally, nucleotide changes that strengthen the hairpin lead neither to uncoupled nor to coupled synthesis of the lysis protein. Structural analysis of mutant MS2 RNA shows that small changes in the stability of the secondary structure lead to substantial differences in translation initiation. The function of the hairpin structure in coupling lysis gene to coat gene translation requires that its stability is kept within narrow limits.

Effect of the sequences upstream from the ribosome-binding site on the yield of protein from the cloned gene for phage MS2 coat protein.

The translational efficiency of the coat protein gene of phage MS2 has been examined in vivo with respect to neighbouring sequences. The cloned MS2 DNA has been gradually shortened starting at the 5' or 3' terminus, and its effect on coat protein synthesis monitored. Removal of the 3'-terminal sequences had little influence. In contrast, the gradual removal of the 5'-terminal region profoundly reduces translation. Long before the ribosomal binding site (RBS) of the coat protein (CP) gene is reached, the yield of CP has dropped by one order of magnitude. Functional half-lives of the various messengers were found not to be significantly different. Available evidence indicates that the secondary structure of the RBS in native and shortened MS2 RNA is identical. We infer that important determinants for ribosome recognition lie 5' to the RBS region of the MS2 RNA coat gene.

Functional recognition of phage RNA by 30-S ribosomal subunits in the absence of initiator tRNA.

30-S ribosomal subunits of Escherichia coli form a stable complex with MS2 RNA or Q beta RNA at 37 degrees C in the absence of initiator tRNA. The complex functions as a precursor of initiation since it can enter the ribosome cycle in the presence of inhibitors of de novo initiation.

Role of 16-S RNA in ribosome messenger recognition.

The deoxyoctanuclotide (5'-3')d(A-A-G-G-A-G-G-T), which is complementary to the 3' end of 16-S RNA, inhibits the formation of the complex between the 30-S subunit and MS2 RNA described in the preceding paper. If the complex is preformed, the octanucleotide cannot prevent entry of the complex into the ribosome cycle upon supplementation with the components for protein synthesis. The subunit . MS2-RNA complex is unable to bind the octanucleotide. It is concluded that in the subunit . phage-RNA initiation precursor the 16-S terminus is base-paired with a complementary MS2 RNA sequence. Edeine, aurintricarboxylic acid and antibodies against ribosomal protein S1 prevent the association of phage RNA with 30-S subunits. These compounds do not, however, inhibit the binding of (5'-3')d(A-A-G-G-A-G-G-T) to 3-S subunits. It is concluded that formation of the complex between MS2 RNA and 30-S subunits does not depend solely on the Shine and Dalgarno base-paring reaction.

Translational fidelity and specificity of ribosomes cleaved by cloacin DF13.

The effect of cloacin DF13 cleavage on several functional properties of the ribosome has been studied in a translational system in vitro. Cleaved ribosomes synthesize relatively shorter polypeptide chains on synthetic and natural templates. Their translational specificity is, however, unchanged as judged by the read-out of MS2 RNA. Here, cleaved as well as control ribosomes start translation only on the coat cistron of the phage RNA. Cloacin cleavage of ribosomes increases their fidelity of translation. Differential inhibition of translation of synthetic and natural template was not observed.