Discriminative effect of rifampin of RNA replication of various RNA bacteriophages.

Rifampin interferes exclusively with RNA replication in vivo of the group I phages MS2, f2, and R17, whereas QbetaRNA replication is unaffected by the drug. In addition, rifampin has a discriminative effect of group I phage RNA replication. In the experimental system employed by us the antibiotic differentially interferes with the synthesis of minus RNA strands in f2, whereas it has almost no effect on the synthesis of progeny plus strands. In MS2, the drug differentially arrests the synthesis of progeny plus strands and almost fails to affect the synthesis of minus RNA strands. In R17 both steps of its RNA replication are affected by rifampin, although each step is only partially (approximately 50%) inhibited. The relation of the present results to the possible role of bacterial proteins and tertiary structure of phage RNA in the process of template recognition is discussed.     

Enzymatic mechanism of RNA translocation in double-stranded RNA bacteriophages

Many complex viruses acquire their genome by active packaging into a viral precursor particle called a procapsid. Packaging is performed by a viral portal complex, which couples ATP hydrolysis to translocation of nucleic acid into the procapsid. The packaging process has been studied for a variety of viruses, but the mechanism of the associated ATPase remains elusive. In this study, the mechanism of RNA translocation in double-stranded RNA bacteriophages is characterized using rapid kinetic analyses. The portal complex of bacteriophage 8 is a hexamer of protein P4, which exhibits nucleotide triphosphatase activity. The kinetics of ATP binding reveals a two-step process: an initial, fast, second-order association, followed by a slower, first-order phase. The slower phase exhibits a high activation energy and has been assigned to a conformational change. ATP binding becomes cooperative in the presence of RNA. Steady-state kinetics of ATP hydrolysis, which proceeds only in the presence of RNA, also exhibits cooperativity. On the other hand, ADP release is fast and RNA-independent. The steady-state rate of hydrolysis increases with the length of the RNA substrate indicating processive translocation. Raman spectroscopy reveals that RNA binds to P4 via the phosphate backbone. The ATP-induced conformational change affects the backbone of the bound RNA but leaves the protein secondary structure unchanged. This is consistent with a model in which cooperativity is induced by an RNA link between subunits of the hexamers and translocation is effected by an axial movement of the subunits relative to one another upon ATP binding.     

A new aspect of the RNA bacteriophages translation control mechanism.

The polarity effect of the coat protein gene of the ribonucleic acid of RNA bacteriophages on the polymerase gene translation will be taken as the basis of the polymerase translation control mechanism. A further condition for this mechanism discussed in this work is the dependence of the phage RNA replication on host cell translation factors. The ribosome binding sites of the phage RNA play a decisive role to realize the control mechanism coding for definite ribosome binding probabilities. The relation between them quantifies the reached polymerase concentration in the early phase of the development of the RNA bacteriophage system in the infected cell.     

Replication of bacteriophages in Escherichia coli mutants thermosensitive in DNA synthesis

Summary In E. coli mutants thermosensitive in DNA synthesis the capacity for replication of bacteriophages , P1 and T4 was studied in order to obtain more information about the biochemical lesions in such strains. Two mutant types were used. In one of them DNA synthesis stops immediately at the restrictive temperature (mutant 165/70). In the other type DNA synthesis continues at the elevated temperature for a residual time period before it comes to a halt (mutant 252). The thermolabile synthetic steps involved in both mutant types are presently still unknown.The temperate phages and P1 differ in their ability to replicate in the mutant types at temperatures non-permissive for host cell DNA synthesis. Replication of phage is blocked in 165/70 but can still take place in 252 after host DNA synthesis has come to a halt. Phage P1 shows the opposite behaviour. It grows in the mutant 165/70 but its ability to replicate in 252 at 42° C is restricted to the period of residual host cell DNA synthesis observed in uninfected cells. Replication of phage T4 on the other hand is unimpeded in both mutants at restrictive temperatures.     

Replication of double-stranded RNA of the virus-like particles in Saccharomyces cerevisiae.

The mode of replication of the L double-stranded RNA (dsRNA) present in virus-like particles in Saccharomyces cerevisiae was examined by density transfer experiments. After transfer to light medium, significant amounts of fully heavy dsRNA persisted over a number of cell doublings. In addition, very little material of hybrid density was ever formed, and the accumulation of fully light material began as early as 0.5 doubling after transfer to light medium. Our results are compatible with a conservative mode of replication or with a semiconservative mode of replication carried out by a small portion of the total dsRNA population. In additional experiments the synthesis of dsRNA relative to the cell cycle was studied. This was done by determining the ratio of short-term to long-term radioactive label in size-separated cell fractions of a prelabeled exponential culture. The ratio of short-term to long-term label remained constant for all fractions, implying that dsRNA is synthesized throughout the cell cycle, increasing through the cell cycle at an exponential rate.     

Subunit contacts of the rifamycin binding site of RNA polymerase (B. subtilis).

The radiolabeled RNA polymerase inhibitors 3-(2-bromoacetamidoethyl)-thiorifamycin and 3-(2-acetamidoethyl)-thiorifamycin quinone have been prepared and covalently attached to the B. subtilis enzyme under mild conditions. Analysis of the subunits labeled indicates that regions of subunits sigma, beta, and beta-prime lie within about 7Å of the 3-position of rifamycin bound to the enzyme, while subunits $\text{alpha}$, beta, and beta-prime lie near the aromatic rings. The results of this work imply that the rifamycin binding site lies near the center of an arrangement involving at least four of the subunits of RNA polymerase. This idea is supported by the results of other studies involving cross-linking of subunits and also rifamycin binding to subunit complexes.     

Control of Replication in RNA Bacteriophages

The rates of viral RNA and protein syntheses for wild-type RNA bacteriophages and their nonpolar, coat protein amber mutants were determined in amber suppressor (S26R1E, Su-1 and H12R8a, Su-3) and nonsuppressor (AB259, S26, and Q13) strains of Escherichia coli in the presence of rifamycin. It was demonstrated that the rates of synthesis of phage-specific replicase and RNA minus strands drop off concurrently in both wild-type and coat protein mutant-infected Su(-) and Su(+) cells after 10 and 15 min postinfection, respectively. The rate of synthesis of RNA plus strands started to decline 5 to 10 min later in both cases. Excessive synthesis of replicase in the coat protein mutant-infected cells was accompanied by a similar overproduction of RNA minus strands, but not of plus strands. Partial suppression of protein synthesis in wild-type phage-infected cells abolishing coat protein control over replicase accumulation led to prolongation of replicase synthesis. Such an effect was observed also in coat protein mutant-infected cells, indicating that the excess of replicase itself may be capable of suppression of replicase synthesis in the absence of coat protein. The prolongation of replicase synthesis was followed by the prolonged synthesis of RNA minus strands in both cases. Moreover, replicase and minus strands were formed in nearly equal amounts when protein synthesis was partially inhibited. Assuming functional instability of phage RNAs, the observed coupling of replicase and minus-strand RNA synthesis offers a possibility for control of viral RNA replication by means of control of replicase synthesis on the translational level. A hypothesis is put forward to explain the molecular mechanism of such coupling between the syntheses of replicase and RNA minus strands.     

Replication of the extrachromosomal ribosomal RNA genes of Tetrahymena thermophilia.

Cultures of Tetrahymena thermophila were deprived of nutrients and later refed with enriched medium to obtain partial synchrony of DNA replication. Preferential replication of the extrachromosomal, macronuclear ribosomal RNA genes (rDNA) was found to occur at 40-80 min after refeeding. The rDNA accounted for one half of the label incorporated into cellular DNA during this period. Electron microscopy of the purified rDNA showed 1% replicative intermediates. Their structure was that expected for bidirectional replication of the linear rDNA from an origin or origins located in the central nontranscribed region of the palindromic molecule. Similar forms had previously been observed for the rDNA of a related species, Tetrahymena pyriformis. The electron microscopic data was consistent with an origin of replication located approximatley 600 base pairs from the center of the rDNA of T. thermophila, in contrast to a more central location in the rDNA of T. pyriformis. One implication of an off-center origin of replication is that there are two such sequences per palindromic molecule.     

Long-range translational coupling in single-stranded RNA bacteriophages: an evolutionary analysis.

In coliphage MS2 RNA a long-distance interaction (LDI) between an internal segment of the upstream coat gene and the start region of the replicase gene prevents initiation of replicase synthesis in the absence of coat gene translation. Elongating ribosomes break up the repressor LDI and thus activate the hidden initiation site. Expression studies on partial MS2 cDNA clones identified base pairing between 1427-1433 and 1738-1744, the so-called Min Jou (MJ) interaction, as the molecular basis for the long-range coupling mechanism. Here, we examine the biological significance of this interaction for the control of replicase gene translation. The LDI was disrupted by mutations in the 3'-side and the evolutionary adaptation was monitored upon phage passaging. Two categories of pseudorevertants emerged. The first type had restored the MJ interaction but not necessarily the native sequence. The pseudorevertants of the second type acquired a compensatory substitution some 80 nt downstream of the MJ interaction that stabilizes an adjacent LDI. In one examined case we confirmed that the second site mutations had restored coat-replicase translational coupling. Our results show the importance of translational control for fitness of the phage. They also reveal that the structure that buries the replicase start extends to structure elements bordering the MJ interaction.