Interactions of Q beta replicase with Q beta RNA

The interactions of Qβ replicase with Qβ RNA were investigated by treating replicase-Qβ RNA complexes under various conditions with ribonuclease T₁, and by characterizing enzyme-bound RNA fragments recovered by a filter binding technique. Evidence for replicase binding at two internal regions of Qβ RNA was obtained. One region (at about 1250 to 1350 nucleotides from the 5′ end) overlaps with the initiation site for coat protein synthesis; this interaction is thought to be inessential for template activity but rather to be involved in the regulation of protein synthesis. Binding to this site (called the S-site) requires moderate concentrations of salt but no magnesium ions. The other region (at about 2550 to 2870 nucleotides from the 5′ end) is probably essential for template activity; binding to this site (called the M-site) is dependent on the presence of magnesium ions. The nucleotide sequences of the RNA fragments from the two sites were determined and found to have no common features. Under the conditions tested, replicase binding at the 3′ end of Qβ RNA could not be demonstrated, except when initiation of RNA synthesis was allowed to occur in the presence of GTP and host factor. If instead of intact Qβ RNA, a complete RNAase T₁ digest of Qβ RNA was allowed to bind to replicase, oligonucleotides from the S-site and the M-site, and oligonucleotides from a region close to the 3′ end, were found to have the highest affinity to the enzyme.The RNA fragments recovered in highest yield, M-2 and S-3 from the M and S-site, respectively, were isolated on a preparative scale and their enzyme binding properties were studied. In competition assays with random RNA fragments of the same size, selective binding was observed both for the M and the S-site fragment. Partial competition for replicase binding was found if M-2 and S-3 were presented simultaneously to the enzyme. Either fragment, if preincubated with replicase, caused a specific inhibition of initiation of Qβ RNA-directed RNA synthesis, without inhibiting the poly(rC)-directed reaction.The results are discussed in terms of a model of replicase-Qβ RNA recognition. Template specificity is attributed to binding of internal RNA regions to replicase, resulting in a specific spatial orientation of the RNA by which the inherently weak, but essential, interaction at the 3′ end is allowed to occur and to lead to the initiation of RNA synthesis.     

Self-catalysed affinity labeling of Q beta replicase.

The spatial neighbourhood of the active center of Q beta replicase can be selectively modified by the method of self-catalysed affinity labeling. In the template-directed, mainly intramolecular enzymatic catalysis, the product [32P]GpG becomes specifically attached to the beta subunit. Using limited digestion of the radioactively labeled polypeptide by cyanogen bromide or N-chlorosuccinimide, we have mapped the attachment site to the region of subunit beta between Trp93 and Met130. Under our reaction conditions, Lys95 is the amino acid most likely to be modified, suggesting that Lys95 lies near the nucleotide binding site in the active center.     

Localization of the Q beta replicase recognition site in MDV-1 RNA

Fragments of MDV-1 RNA (a small, naturally occurring template for Q beta replicase) that were missing nucleotides at either their 5' end or their 3' end were still able to form a complex with Q beta replicase. By assaying the binding ability of fragments of different length, it was established that the binding site for Q beta replicase is determined by nucleotide sequences that are located near the middle of MDV-1 RNA. Fragments missing nucleotides at their 5' end were able to serve as templates for the synthesis of complementary strands, but fragments missing nucleotides at their 3' end were inactive, indicating that the 3'-terminal region of the template is required for the initiation of RNA synthesis. The nucleotide sequences of both the 3' terminus and the central binding region of MDV-1 (+) RNA are almost identical to sequences at the 3' terminus and at an internal region of Q beta (-) RNA.     

Segmental flexibility of ribosomal protein S1 bound to ribosomes and Q beta-replicase

The protonization pattern of the endogenous donor component D₁ which feeds electrons directly into chl-a⁺_II has been analyzed in Tris-washed inside-out thylakoids with the aid of appropriate pH-indicators. It was found that under repetitive flash excitation the amount of protons released is proportional to the extent of D₁-oxidation, depending on the time between the flashes. The kinetics of D₁-oxidation (being practically the same as in normal Tris-washed chloroplasts) are faster than the proton release by two orders of magnitude. The results lead to the conclusion that D₁ is protonized in the reduced state with pK(D^ox₁) < 5 and becomes deprotonized in the oxidized state with pK(D^red₁) ? 8. The proton release is kinetically limited by a transport barrier. Implications on the interpretation of the proton release pattern in preparation with intact water oxidation are discussed.     

Function of bacteriophage Qbeta replicase containing an altered subunit IV

In order to elucidate the function of elongation factor Ts in Qβ replicase, enzyme was obtained from a Qβ-infected Escherichia coli mutant HAK88, which carries an altered EFTs² with a thermolabile catalytic activity. HAK88 Qβ replicase was found to be quite unstable at 42 °C. Further studies indicated that the mutant enzyme exhibits temperature sensitivity with regard to GTP binding ability but not with Qβ RNA and poly(C) binding. These results suggest that the function of EFTs in Qβ replicase is closely related to the binding of GTP to the enzyme.A defect in Qβ replicase also appears when it is reconstituted from the Qβ replicase subunit complex I–II and the HAK88 EFTu-EFTs complex. Several lines of evidence obtained by using the reconstituted enzyme suggest strongly that the EFTs function is involved specifically in initiation of RNA synthesis, but not in the elongation reaction.     

Structural consensus of RNA templates replicated by Q beta replicase.

Q beta replicase replicates a variety of enzyme-specific small RNAs in addition to the phage genomic RNA. The sequence analysis has revealed that all these RNAs are potentially capable of forming a consensus secondary structure element. It represents a stalk which is formed by the 5'-GGG ... and ... CCCA-3' complementary stretches at the termini of the replicating RNA molecules and adjacent 5'- and 3'-hairpins, which may form a stacking with the stalk. The structure found is rather similar to the analogous structure in the tRNA molecule. The genomic RNA of the Q beta phage and other related phages can also form a similar structural element.     

Terminal adenylation in the synthesis of RNA by Q beta replicase

We investigated the apparent requirement that Q beta replicase must add a nontemplated adenosine to the 3' end of newly synthesized RNA strands. We used abbreviated MDV-1 (+)-RNA templates that lacked either 62 or 63 nucleotides at their 5' end in Q beta replicase reactions. The MDV-1 (-)-RNA strands synthesized from these abbreviated (+)-strand templates were released from the replication complex, yet they did not possess a nontemplated 3'-terminal adenosine. These results imply that, despite observations that all naturally occurring RNAs synthesized by Q beta replicase possess a nontemplated 3'-adenosine, the addition of an extra adenosine is not an obligate step for the release of completed strands. Since the abbreviated templates lacked a normal 5' end, it is probable that a particular sequence at the 5' end of the template is required for terminal adenylation to occur.     

On the nature of spontaneous RNA synthesis by Q beta replicase.

Numerous RNA species of different length and nucleotide sequence grow spontaneously in vitro in Q beta replicase reactions where no RNA templates are added deliberately. Here, we show that this spontaneous RNA synthesis by Q beta replicase is template directed. The immediate source of template RNA can be the laboratory air, but there are ways to eliminate, or at least substantially reduce, the harmful effects of spontaneous synthesis. Solitary RNA molecules were detected in a thin layer of agarose gel containing Q beta replicase, where they grew to form colonies that became visible upon staining with ethidium bromide. This result provides a powerful tool for RNA cloning and selection in vitro. We also show that replicating RNAs similar to those growing spontaneously are incorporated into Q beta phage particles and can propagate in vivo for a number of phage generations. These RNAs are the smallest known molecular parasites, and in many aspects they resemble both the defective interfering genomes of animal and plant viruses and plant virus satellite RNAs.     

mRNA containing an extended Shine-Dalgarno sequence is translated independently of ribosomal protein S1.

The role of ribosomal protein S1 in the translation of mRNA containing an extended Shine-Dalgarno sequence was investigated. Using the toeprinting technique, formation of the ternary initiation complex between 30S subunits, both S1-depleted or treated with anti-S1 antibodies, and mini-mRNA containing the 9 nucleotide-long Shine-Dalgarno sequence was studied. It was concluded that the initiation of translation on mRNA with an extended Shine-Dalgarno sequence is S1-independent. It was demonstrated that S1-depleted ribosomes effectively translate the cro-mini-mRNA in a cell-free system. In contrast to cro-mini-mRNA, 30S subunits without protein S1 are inactive in ternary initiation complex formation with, and cell-free translation of, MS2 or fr phage RNAs and RNA protein III of phage fd.     

Kinetic analysis of template-instructed and de novo RNA synthesis by Q beta replicase

The kinetics of template-free and template-instructed RNA synthesis by Q_β replicase were investigated. Template-instructed RNA synthesis has different growth rates in the exponential (excess enzyme) and the linear (excess template) phase of growth. In the absence of exogenous template, Q_β replicase synthesizes self-replicating RNA after an initial lag phase (“template-free” synthesis). The lag time can be determined by extrapolating the growth curve to the time of appearance of the first self-replicating strand. Growth rates in the exponential and linear phase, and especially the times of the lag phase for nucleotide incorporations under identical template-free conditions, show considerable scattering in contrast to the deterministic behavior of template-instructed synthesis. Evaluation of the kinetic data reveals that the time lag of template-free synthesis is strongly dependent on the concentration of the nucleoside triphosphate and the enzyme. The lag time is approximately inversely proportional to the powers 2.75 of the nucleotide and 2.5 of the enzyme concentration, respectively, both being lower limit values. The rate of template-instructed RNA synthesis is linearly proportional to the enzyme concentration and less than linearly proportional to the triphosphate concentration, in accordance with a substrate dependence of a Michaelis-Menten type of mechanism. The kinetic data cannot be reconciled with the proposition that template-free synthesis is due to low concentrations of templates present as impurities in the incorporation mixture and giving rise to autocatalytic RNA synthesis by a template-instructed mechanism. The data strongly favor the de novo mechanism proposed by Sumper &; Luce (1975).     

Q beta replicase: mapping the functional domains of an RNA-dependent RNA polymerase

We have localized a functional region of the RNA bacteriophage Q beta replicase following an extensive mutational analysis. Using the method of oligonucleotide linker-insertion mutagenesis, we specifically introduced mutations into a cloned DNA copy of the Q beta replicase gene so that the resulting replicase products would putatively contain small amino acid insertions. In a selective phenotypic assay, we screened mutant replicases for RNA-directed replication activity in vivo. Analysis of 37 different mutant clones indicated that Q beta replicase can accept amino acid substitutions and insertions at several sites at the amino and carboxy termini without abolishing functional activity in vivo or in vitro. However, disruption within the internal amino acid sequence resulted almost exclusively in nonfunctional enzyme. The results suggest that the central region of the replicase protein contains a rigid amino acid composition that is required for replicase function, whereas the amino and carboxy termini are much more receptive to small amino acid insertions and substitutions. These experiments should further enable us to analyze the coding function of the Q beta replicase gene independently of other phage RNA functions contained within this nucleotide region.     

In vitro recombination and terminal elongation of RNA by Q beta replicase.

SV-11 is a short-chain [115 nucleotides (nt)] RNA species that is replicated by Q beta replicase. It is reproducibly selected when MNV-11, another 87 nt RNA species, is extensively amplified by Q beta replicase at high ionic strength and long incubation times. Comparing the sequences of the two species reveals that SV-11 contains an inverse duplication of the high-melting domain of MNV-11. SV-11 is thus a recombinant between the plus and minus strands of MNV-11 resulting in a nearly palindromic sequence. During chain elongation in replication, the chain folds consecutively to a metastable secondary structure of the RNA, which can rearrange spontaneously to a more stable hairpin-form RNA. While the metastable form is an excellent template for Q beta replicase, the stable RNA is unable to serve as template. When initiation of a new chain is suppressed by replacing GTP in the replication mixture by ITP, Q beta replicase adds nucleotides to the 3' terminus of RNA. The replicase uses parts of the RNA sequence, preferentially the 3' terminal part for copying, thereby creating an interior duplication. This reaction is about five orders of magnitude slower than normal template-instructed synthesis. The reaction also adds nucleotides to the 3' terminus of some RNA molecules that are unable to serve as templates for Q beta replicase.     

The conformational properties of ribosomal protein S1.

The proton NMR spectrum of S1 reveals that S1 has considerable tertiary structure in physiological buffers, but more structural flexibility than normal for globular proteins. S1's NMR spectrum is independent of the method of preparation.     

An Escherichia coli K12 mutant carrying altered ribosomal protein (S10).

An $\text{Escherichia coli}$ mutant (JE14373) carrying decreased stability of stable RNA species was found to have altered electrophoretic mobility of a 30S ribosomal protein (S10). Recombinants covering $\text{str}$ gene (76 min on $\text{E. coli}$ linkage map by Bachmann, Low and Taylor, 1976 (ref. 1)) obtained from a cross of CSH64 × JE14373, restored normal S10 protein. The size analysis of RNAs labeled for 15 min with [³H]uridine showed 50 to 60 % decrease of 16S RNA in this mutant strain, but almost no decrease of 23S RNA at 10 or 40 min after addition of rifampicin. On the other hand, no change was observed in the stability of both rRNA pieces in its parental PA3092 strain even at 40 min after addition of rifampicin.