Qβ replicase: Mapping the functional domains of an RNA-dependent RNA polymerase

Mills, Donald R.; Priano, Christine; DiMauro, Paola; Binderow, B D

doi:10.1016/0022-2836(89)90319-7

Cited by 30 publications

(36 citation statements)

References 28 publications

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“…3). These results, coupled to the observation that the amino acid sequence of the viral polypeptide is similar to known polymerases (20) 11562 Biochemistry: Brown and Gold by Q0B replicase (22). This would have the same effect as binding near the terminus, as host factor localizes the 3' end of the template to an area where Q,B replicase is bound.…”

Section: Resultsmentioning

confidence: 67%

RNA replication by Q beta replicase: a working model.

Brown

Gold

1996

Proc. Natl. Acad. Sci. U.S.A.

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Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Qj8 replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I-and site 1I-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Qf3 replicase.Q03 replicase is the RNA-dependent RNA polymerase responsible for replicating the single-stranded RNA genome of the coliphage Q0 (1). The replicase is a heterotetramer comprising a virally encoded subunit (2, 3) plus three host proteins: ribosomal protein S1 (4, 5) and elongation factors Tu (EF-Tu) and Ts (6). Q03 replicase is remarkable in three respects: (i) it effects a 10,000-fold amplification of the 4200-nucleotide single-stranded RNA of Q0( during the very short infection time of the phage (7), (ii) it specifically replicates the viral genomic RNA in the presence of a vast excess of host RNA, and (iii) it copies entire template RNAs, from 3' terminus to 5' terminus, without utilizing endogenous primers.The first feat is accomplished by exponential amplification (8). The polymerase uses the plus strand of the phage genome as a template to produce a complementary RNA, referred to as the minus strand. Both the plus and minus strands serve as templates for the replicase, thus the amount of phage RNA doubles with each replication cycle. The molecular mechanism by which the enzyme achieves template recognition and initiates complementary strand synthesis at the 3' end of the RNA, however, has yet to be resolved. In a previous report (9), SELEX (systematic evolution of ligands by exponential enrichment) was used to generate two families of RNA ligands (referred to as class I and class II) that bound Q0 replicase with nanomolar dissocation constants.The class I ligands possessed single-stranded regions containing A's and C's that were essential for binding the replicase. A region of the plus strand of the Q0( genome that is required for replication, and that binds specifically to Q0( replicase, possesses a similar sequence motif (10, 11). The class II RNAs had polypyrimidine tracts that were required for high affinity binding to the replicase. The Q03 genomic minus strand, as well as most of the small replicated RNAs identified by in vitro experiments, possess similar pyrimidine rich regions (9). Importantly, the two classes of RNAs did not compete for binding to the replicase, indicating that two independent binding sites exist on the phage polymerase. The two regions of Q03 replicase that ar...

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Section: Resultsmentioning

confidence: 67%

RNA replication by Q beta replicase: a working model.

Brown

Gold

1996

Proc. Natl. Acad. Sci. U.S.A.

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“…(24), all resulting in decreased or temperature-sensitive activity. One study of QJ3 replicase found that insertions in most parts of the molecule inactivate it (39). In the studies of poliovirus (23), polymerase was produced in Escherichia coli in soluble form and the mutants produced were examined for some of their enzymatic properties.…”

Section: Consensus Conformation: H H H H H H H H H H E E E E T T T E mentioning

confidence: 99%

RNA-dependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus: definition of essential domains.

Ribas

Wickner²

1992

Proc. Natl. Acad. Sci. U.S.A.

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The L-A double-stranded RNA virus of Saccharomyces cerevisiac makes a gag-pol fusion protein by a -1 ribosomal frameshift. The pol amino acid sequence includes consensus patterns typical of the RNA-dependent RNA polymerases (EC 2.7.7.48) of (+) strand and double-stranded RNA viruses of animals and plants. We have carried out "alaninescanning mutagenesis" of the region of L-A including the two most conserved polymerase motifs, SG...T...NT..N (. = any amino acid) and GDD. By constructing and analyzing 46 different mutations in and around the RNA polymerase consensus regions, we have precisely dermed the extent of domains and specific residues essential for viral replication. Assuming that this highly conserved region has a common secondary structure among different viruses, we predict a largely fl-sheet structure.The (+) strand RNA viruses of animals and plants share amino acid sequence patterns in their RNA-dependent RNA polymerase (EC 2.7.7.48) regions (1-3) (Fig. 1). The same patterns are present in most of the dsRNA viruses whose polymerase segment has been sequenced, including the L-A virus of Saccharomyces cerevisiae (4), reovirus (5), bluetongue virus (6), and rotavirus (7). Two other dsRNA viruses [46 phage (8) and infectious bursal disease virus (9)] show a less clear fit to the pattern. The extent of these common sequence patterns suggests that there is a common structure and function among the more than 50 viral enzymes for which sequence data are available and that information obtained about one of them may be applicable to others. While different enzymes must recognize different sites on viral RNA and interact with different viral and host proteins, there are also common functions which they must carry out, including binding of Mg2' and rNTPs, holding onto the template RNA chain, and the chain elongation reaction itself. It is likely that these conserved sequence patterns are part of domains responsible for such common functions. The L-A dsRNA virus of yeast replicates by a conservative mechanism with (+) and (-) strands made inside the viral particle at different points in the replication cycle (reviewed in ref. 10). In vitro systems for replication, transcription, and packaging are available, and the signals for the replication step and for packaging have been defined (11)(12)(13)(14). L-A encodes its 70-kDa major coat protein (called gag) (4, 15) and a 170-kDa gag-pol fusion protein (4, 16) formed by a -1 ribosomal frameshift indistinguishable in mechanism from that used by retroviruses for a similar purpose (17). The pol region of the gag-pol fusion protein (4) has all the characteristic sequence patterns identified by Kamer and Argos (1) as typical of (+) strand RNA viral RNA-dependent RNA poly- We have defined the regions surrounding the two most highly conserved RNA polymerase consensus patterns that are necessary for viral propagation. We show that, although these domains are highly conserved among a broad range of viruses in both primary structure and predicted secondary structure, t...

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“…The sequences of the replicases of the related small RNA bacteriophages from E. coli, designated as MS2, SP and GA, have also been determined 137 -391. Similarities between the replicase protein sequences of the different RNA phages were found to be clustered around the center of the peptide chains [40]. It is interesting to note that the neighbourhood surrounding the attachment site for the nucleotide analog at Lys95 does not show sequence similarity to the related RNA phages.…”

Section: Discussionmentioning

confidence: 89%

Self‐catalysed affinity labeling of Qβ replicase

Lindner

Glaser

Biebricher

et al. 1991

European Journal of Biochemistry

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The spatial neighbourhood of the active center of QP 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 /3 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 between Trp93 and Metl30. 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.Self-catalysed affinity labeling allows the selective marking of an amino acid residue in the spatial neighbourhood of the active center of enzymes. This has been demonstrated for a large number of viral, eubacterial, archaebacterial and eukaryotic DNA-directed RNA polymerases with widely differing subunit composition [1 -91. The labeling results from two separate but consecutive reactions. In the first step, the RNA polymerase protein reacts chemically with a nucleotide which has been modified by a reactive group such as an aldehyde and this is followed by treatment with borohydride. The subsequent enzymatic step occurs when template and ribonucleoside [~x-~~Pltriphosphate are added. If the catalytic activity of the polymerase is not destroyed by the chemical modification in the first step, then a radioactive product covalently bound to the protein may be formed. Formation of this product depends on whether the attachment site of the bound nucleotide analog is close enough to the active center of the polymerase to enable the nucleotide to reach into the binding site of the enzyme. The successful application of this self-catalysed labeling to rather different DNA-directed polymerases demonstrates that both of these conditions are met by this class of enzymes. In contrast to DNA-directed polymerases, Qp replicase is an RNA-directed RNA polymerase. Abbreviation. M D V -1 (+)RNA, midivariant-1 R N A plus strand. Enzymes. DNA-directed RNA polymerase, nucleoside-triphosphate:RNA nucleotidykransferdse (DNA-directed) (EC 2.7.7.6); alkaline phosphatase, orthophosphoric-monoester phosphohydratase (alkaline optimum) (EC 3.1.3.1); nucleotide pyrophosphatase, dinucleotide nucleotidohydrolase (EC 3.6.1 .Y); ribonuclease T2 (EC 3.1.27.1); Tritirachium alkaline proteinase, proteinase K (EC 3.4.21.14); Myxobacter AL-1 proteinase, endoproteinase Lys-C (EC 3.4.99.30).Subunit M is only required for replication of the Qp plus strand [14,15]. In vitro and at low template concentrations, QP replicase requires a highly specific RNA with an oligo(C) tail at its 3'-end [16]. In this paper, we show that the method of self-catalysed affinity labeling can be successfully applied to an RNA-directed polymerase such as QB replicase. A preliminary report of these investigations has been published [8]. Recently this method was also used to label other RNA-directed RNA polymerases such as the replicase of tick-borne e...

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Qβ replicase: Mapping the functional domains of an RNA-dependent RNA polymerase

Cited by 30 publications

References 28 publications

RNA replication by Q beta replicase: a working model.

RNA replication by Q beta replicase: a working model.

RNA-dependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus: definition of essential domains.

Self‐catalysed affinity labeling of Qβ replicase

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