Different Mechanisms of Recognition of Bacteriophage Qβ Plus and Minus Strand RNAs by Qβ Replicase

Barrera, Imma; Schuppli, Daniel; Sogo, José M.; Weber, Hans

doi:10.1006/jmbi.1993.1407

Cited by 102 publications

(121 citation statements)

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“…Thus, the intercistronic region may not be absolutely required for (-)-strand initiation but may greatly increase the frequency of initiation. The possibility that some factor(s) interacts with intercistronic sequences to facilitate initiation at the 3' end of BMV RNA3 is'similar to proposals that Q,B RNA replicase binds an internal M site on Q,B RNA and remains attached there while initiating (-)-strand synthesis from the 3' end (24) and to the role of an internal enhancer in L-A virus (-)-strand synthesis (25). Similarly, template RNA sequences distal to coronavirus transcription initiation sites may recruit cell and viral factors into a functioning transcription complex…”

Section: Methodsmentioning

confidence: 96%

Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

Quadt

Ishikawa

Janda

et al. 1995

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

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In this report we show that yeast expressing brome mosaic virus (BMV) replication proteins la and 2a and replicating a BMV RNA3 derivative can be extracted to yield a template-dependent BMV RNA-dependent RNA polymerase (RdRp) able to synthesize (-)-strand RNA from BMV (+)-strand RNA templates added in vitro. This virus-specific yeast-derived RdRp mirrored the template selectivity and other characteristics of RdRp from BMV-infected plants. Equivalent extracts from yeast expressing la and 2a but lacking RNA3 contained normal amounts of la and 2a but had no RdRp activity on BMV RNAs added in vitro. To determine which RNA3 sequences were required in vivo to yield RdRp activity, we tested deletions throughout RNA3, including the 5', 3', and intercistronic noncoding regions, which contain the cis-acting elements required for RNA3 replication in vivo. RdRp activity was obtained only from cells expressing la, 2a, and RNA3 derivatives retaining both 3' and intercistronic noncoding sequences. Strong correlation between extracted RdRp activity and BMV (-)-strand RNA accumulation in vivo was found for all RNA3 derivatives tested. Thus, extractable in vitro RdRp activity paralleled formation of a complex capable ofviral RNA synthesis in vivo. The results suggest that assembly of active RdRp requires not only viral proteins but also viral RNA, either to directly contribute some nontemplate function or to recruit essential host factors into the RdRp complex and that sequences at both the 3'-terminal initiation site and distant internal sites of RNA3 templates may participate in RdRp assembly and initiation of (-)-strand synthesis.Brome mosaic virus (BMV), a plant-infecting virus, is a representative member of the alphavirus-like superfamily of (+)-strand RNA viruses of animals and plants. The BMV genome consists of three separately encapsidated RNAs, designated RNA1-3. Monocistronic RNA1 and RNA2, respectively, encode proteins la (109 kDa) and 2a (94 kDa). la and 2a are essential mutually interacting components of the RNA-dependent RNA polymerase (RdRp) involved in BMV RNA replication (1, 2). This RdRp, whose functions have been studied in vivo and in vitro, is a complex of la, 2a, and host proteins (2-6). RNA3 contains two genes separated by an -250-nt intercistronic region. The 5'-proximal gene, encoding the 3a infection movement protein (7,8), is translated directly from RNA3. The 3'-proximal coat protein gene is expressed via a subgenomic mRNA, RNA4. The 3a and coat proteins are dispensable for RNA replication but are required for infection spread in plants (8).BMV-directed replication of RNA3 in vivo depends on cis-acting sequences in three regions of RNA3 (see Fig. 3A): the 3' and 5' noncoding regions (NCRs) and the intercistronic NCR (9). The last 160-200 nt of the 3' NCR contain signals for initiation of (-)-strand RNA synthesis in vitro and areThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U....

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Section: Methodsmentioning

confidence: 96%

Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

Quadt

Ishikawa

Janda

et al. 1995

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

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“…We believe that the specificity mechanism we have described may be used rather commonly in the replication of positive strand RNA viruses+ It fits observations made in mapping the genomic promoters recognized by turnip crinkle virus (TCV) RdRp in both the positive and negative sense strands of TCVassociated satellite RNA C (Song & Simon, 1995;Guan et al+, 1997), and may also apply to subgenomic RNA synthesis from this satellite RNA (Wang & Simon, 1997)+ It may apply to the RNAs replicated by Qb replicase: the well-known requirement for secondary structure might not only be in order to ensure product release from the template strands (Arora et al+, 1996), but perhaps also an expression of the specificity mechanism we have described+ Interestingly, although potential promoter elements have been described on Qb RNA, Barrera et al+ (1993) have suggested that "template selectivity may be provided by the tertiary structure and topology of the RNA, and in addition by the C-rich 39-terminal sequence+" Further, the observation that poliovirus and human rhinovirus genomes entirely lacking the 39-noncoding region upstream of the poly(A) tail (deletions of 68 and 44 nt, respectively) were capable of replication (albeit debilitated) led to the conclusion that the basic mechanism of picornavirus replication initiation may not be strictly template specific (Todd et al+, 1997)+ The observed replication of the truncated genomes might alternatively be explained in terms of a specificity mechanism like that used by TYMV RdRp+ Finally, the template preferences we have observed for TYMV RdRp are remarkably similar to the template requirements of the Mauriceville retroplasmid reverse transcriptase (Chen & Lambowitz, 1997), an enzyme that has been considered to be evolutionarily intermediate between TYMV-like RdRp's and retroviral-like reverse transcriptases (Maizels & Weiner, 1994)+ These observations suggest that it will be useful to consider the specificity principle we have described here in future searches for cis-active elements controlling RNA transcription in positive strand viruses+ We believe that this mechanism may also be found to operate in other RNA-protein interactions, such as transcript binding by the bacterial rho termination protein, for which RNA recognition criteria have proven elusive (Platt, 1994), or in the selection of splice sites in precursor mRNAs (Goguel & Rosbash, 1993;Black, 1995)+…”

Section: Applicability Of This Specificity Mechanism To Other Proteinmentioning

confidence: 95%

Specific site selection in RNA resulting from a combination of nonspecific secondary structure and -CCR- boxes: Initiation of minus strand synthesis by turnip yellow mosaic virus RNA-dependent RNA polymerase

Singh

Dreher

1998

RNA

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A turnip yellow mosaic virus RNA-dependent RNA polymerase activity was used to study the template requirements for in vitro minus strand synthesis, which is initiated specifically opposite the 39-CCA that terminates the 39-tRNA-like structure. A deletion survey confirmed earlier results suggesting the absence of minus strand promoter elements upstream of the pseudoknotted acceptor stem and 39-terminus. Reiteration of this 27-nt domain provided two competing initiation sites. By varying the added downstream element, it was shown that the pseudoknotted domain could be functionally replaced by various simple stem/loops, although with some decrease in activity. The addition of varying numbers of consecutive -CCA-triplets to the 39 end of the tRNA-like structure resulted in accurate initiation from each added triplet. A similar spectrum of initiations occurred with an unstructured RNA consisting of 12 consecutive -CCA-triplets and no additional viral sequence. Substitution mutations revealed no influence on minus strand synthesis of the identity of the nucleotide immediately upstream of a -CC-initiation site, but a preference for a purine immediately downstream. The introduction of secondary structure into the linear template showed that the usage of potential -CCR-initiation sites is influenced by nonspecific secondary structure. We conclude that specificity arises from the requirement that a -CCR-sequence be sterically accessible. This mechanism is only applicable to interactions that do not involve RNA unwinding during site selection, but may be used commonly in positive strand RNA virus replication and be applicable to other RNA-protein interactions.

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“…Hfq was originally identified as a host factor in Escherichia coli for the replication of the Qβ phage (3), where it binds to the C-rich 3′ end of plus-strand viral RNA (4). Subsequently, physiological roles of Hfq were described in the regulation of mRNA translation and in mRNA degradation, where it modulates the processing of RNA 3′ ends (1,(5)(6)(7)(8).…”

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confidence: 99%

Structural basis for RNA 3′-end recognition by Hfq

Sauer

Weichenrieder

2011

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

192

251

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The homohexameric (L)Sm protein Hfq is a central mediator of small RNA-based gene regulation in bacteria. Hfq recognizes small regulatory RNAs (sRNAs) specifically, despite their structural diversity. This specificity could not be explained by previously described RNA-binding modes of Hfq. Here we present a distinct and preferred mode of Hfq-RNA interaction that involves the direct recognition of a uridine-rich RNA 3′ end. This feature is common in bacterial RNA transcripts as a consequence of Rho-independent transcription termination and hence likely contributes significantly to the general recognition of sRNAs by Hfq. Isothermal titration calorimetry shows nanomolar affinity between Salmonella typhimurium Hfq and a hexauridine substrate. We determined a crystal structure of the complex that reveals a constricted RNA backbone conformation in the proximal RNA-binding site of Hfq, allowing for a direct protein contact of the 3′ hydroxyl group. A free 3′ hydroxyl group is crucial for the high-affinity interaction with Hfq also in the context of a full-length sRNA substrate, RybB. The capacity of Hfq to occupy and sequester the RNA 3′ end has important implications for the mechanisms by which Hfq is thought to affect sRNA stability, turnover, and regulation.RNA chaperone | regulation of translation | RNA degradation | prokaryotes H fq is an abundant and widely conserved RNA-binding protein in bacteria and a major player in the RNA-based regulation of gene expression, such as in the adaptive response to cell stress or in the induction of virulence (1, 2).Hfq was originally identified as a host factor in Escherichia coli for the replication of the Qβ phage (3), where it binds to the C-rich 3′ end of plus-strand viral RNA (4). Subsequently, physiological roles of Hfq were described in the regulation of mRNA translation and in mRNA degradation, where it modulates the processing of RNA 3′ ends (1,(5)(6)(7)(8). The most prominent function of Hfq, however, is its interaction with small regulatory RNAs (sRNAs) that act in trans and that are differentially expressed under various metabolic and environmental conditions (9, 10). They globally regulate gene expression via base-pairing to frequently entire sets of partially complementary mRNAs (11,12). Hfq stabilizes sRNAs in the absence of their targets (13). Hfq was also found to facilitate base-pairing to the mRNAs and help trigger subsequent steps, such as the repression of translation and/or the acceleration of decay, but also mRNA activation (11,14). Despite their structural diversity, the recognition of many sRNAs by Hfq is highly specific and even works across species barriers (15). It is an intriguing question how this selectivity is achieved.Crystal structures reveal that bacterial Hfq adopts an (L)Sm fold and forms homohexameric rings, whereas related (L)Sm proteins [Sm proteins and Sm-like (LSm) proteins] in archaea and eukaryotes are found to form homomeric or heteromeric heptamers, respectively (16). Two distinct RNA-binding sites have been described on opposite f...

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Different Mechanisms of Recognition of Bacteriophage Qβ Plus and Minus Strand RNAs by Qβ Replicase

Cited by 102 publications

References 0 publications

Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

Specific site selection in RNA resulting from a combination of nonspecific secondary structure and -CCR- boxes: Initiation of minus strand synthesis by turnip yellow mosaic virus RNA-dependent RNA polymerase

Structural basis for RNA 3′-end recognition by Hfq

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