Skip to main content
PMC home page
RNA logoLink to RNA
. 2000 May;6(5):698–707. doi: 10.1017/s1355838200992410

CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases.

S Yoshinari 1, P D Nagy 1, A E Simon 1, T W Dreher 1
PMCID: PMC1369950  PMID: 10836791

Abstract

It has previously been observed that the only specific requirement for transcriptional initiation on viral RNA in vitro by the RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus is the CCA at the 3' end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdRp, and Qbeta replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multiple CCA boxes but lacking specific viral sequences. Each enzyme is able to initiate transcription from several CCA boxes within these RNAs, and no special reaction conditions are required for these activities. The transcriptional yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redundant sequences presents a challenge to the specificity of viral transcription and replication. We identify 3'-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit initiation among potential CCA initiation sites. These two specificity mechanisms are used to different degrees by the three RdRps. The finding that three viral RdRps representing two of the three supergroups within the positive-strand RNA viral RdRp phylogeny support substantial transcription in the absence of unique promoters suggests that this phenomenon may be common among positive-strand viruses. A framework is presented arguing that replication of viral RNA in the absence of unique promoter elements is feasible.

Full Text

The Full Text of this article is available as a PDF (440.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ball L. A. Replication of the genomic RNA of a positive-strand RNA animal virus from negative-sense transcripts. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12443–12447. doi: 10.1073/pnas.91.26.12443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bausch J. N., Kramer F. R., Miele E. A., Dobkin C., Mills D. R. Terminal adenylation in the synthesis of RNA by Q beta replicase. J Biol Chem. 1983 Feb 10;258(3):1978–1984. [PubMed] [Google Scholar]
  3. Blumenthal T., Carmichael G. G. RNA replication: function and structure of Qbeta-replicase. Annu Rev Biochem. 1979;48:525–548. doi: 10.1146/annurev.bi.48.070179.002521. [DOI] [PubMed] [Google Scholar]
  4. Bolten R., Egger D., Gosert R., Schaub G., Landmann L., Bienz K. Intracellular localization of poliovirus plus- and minus-strand RNA visualized by strand-specific fluorescent In situ hybridization. J Virol. 1998 Nov;72(11):8578–8585. doi: 10.1128/jvi.72.11.8578-8585.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown D., Gold L. RNA replication by Q beta replicase: a working model. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11558–11562. doi: 10.1073/pnas.93.21.11558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carpenter C. D., Simon A. E. Analysis of sequences and predicted structures required for viral satellite RNA accumulation by in vivo genetic selection. Nucleic Acids Res. 1998 May 15;26(10):2426–2432. doi: 10.1093/nar/26.10.2426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chapman M. R., Kao C. C. A minimal RNA promoter for minus-strand RNA synthesis by the brome mosaic virus polymerase complex. J Mol Biol. 1999 Feb 26;286(3):709–720. doi: 10.1006/jmbi.1998.2503. [DOI] [PubMed] [Google Scholar]
  8. Deiman B. A., Koenen A. K., Verlaan P. W., Pleij C. W. Minimal template requirements for initiation of minus-strand synthesis in vitro by the RNA-dependent RNA polymerase of turnip yellow mosaic virus. J Virol. 1998 May;72(5):3965–3972. doi: 10.1128/jvi.72.5.3965-3972.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dreher T. W., Bujarski J. J., Hall T. C. Mutant viral RNAs synthesized in vitro show altered aminoacylation and replicase template activities. Nature. 1984 Sep 13;311(5982):171–175. doi: 10.1038/311171a0. [DOI] [PubMed] [Google Scholar]
  10. Guan H., Song C., Simon A. E. RNA promoters located on (-)-strands of a subviral RNA associated with turnip crinkle virus. RNA. 1997 Dec;3(12):1401–1412. [PMC free article] [PubMed] [Google Scholar]
  11. Hardy S. F., German T. L., Loesch-Fries L. S., Hall T. C. Highly active template-specific RNA-dependent RNA polymerase from barley leaves infected with brome mosaic virus. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4956–4960. doi: 10.1073/pnas.76.10.4956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Koonin E. V., Dolja V. V. Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol. 1993;28(5):375–430. doi: 10.3109/10409239309078440. [DOI] [PubMed] [Google Scholar]
  13. Kuchino Y., Nishimura S. Enzymatic RNA sequencing. Methods Enzymol. 1989;180:154–163. doi: 10.1016/0076-6879(89)80099-0. [DOI] [PubMed] [Google Scholar]
  14. Lai M. M. Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology. 1998 Apr 25;244(1):1–12. doi: 10.1006/viro.1998.9098. [DOI] [PubMed] [Google Scholar]
  15. Li T., Ho H. H., Maslak M., Schick C., Martin C. T. Major groove recognition elements in the middle of the T7 RNA polymerase promoter. Biochemistry. 1996 Mar 26;35(12):3722–3727. doi: 10.1021/bi9524373. [DOI] [PubMed] [Google Scholar]
  16. Meyer F., Weber H., Weissmann C. Interactions of Q beta replicase with Q beta RNA. J Mol Biol. 1981 Dec 15;153(3):631–660. doi: 10.1016/0022-2836(81)90411-3. [DOI] [PubMed] [Google Scholar]
  17. Miller W. A., Bujarski J. J., Dreher T. W., Hall T. C. Minus-strand initiation by brome mosaic virus replicase within the 3' tRNA-like structure of native and modified RNA templates. J Mol Biol. 1986 Feb 20;187(4):537–546. doi: 10.1016/0022-2836(86)90332-3. [DOI] [PubMed] [Google Scholar]
  18. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miranda G., Schuppli D., Barrera I., Hausherr C., Sogo J. M., Weber H. Recognition of bacteriophage Qbeta plus strand RNA as a template by Qbeta replicase: role of RNA interactions mediated by ribosomal proteins S1 and host factor. J Mol Biol. 1997 Apr 18;267(5):1089–1103. doi: 10.1006/jmbi.1997.0939. [DOI] [PubMed] [Google Scholar]
  20. Moody M. D., Burg J. L., DiFrancesco R., Lovern D., Stanick W., Lin-Goerke J., Mahdavi K., Wu Y., Farrell M. P. Evolution of host cell RNA into efficient template RNA by Q beta replicase: the origin of RNA in untemplated reactions. Biochemistry. 1994 Nov 22;33(46):13836–13847. doi: 10.1021/bi00250a038. [DOI] [PubMed] [Google Scholar]
  21. Nagy P. D., Pogany J., Simon A. E. RNA elements required for RNA recombination function as replication enhancers in vitro and in vivo in a plus-strand RNA virus. EMBO J. 1999 Oct 15;18(20):5653–5665. doi: 10.1093/emboj/18.20.5653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Negrutskii B. S., Stapulionis R., Deutscher M. P. Supramolecular organization of the mammalian translation system. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):964–968. doi: 10.1073/pnas.91.3.964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nishihara T., Mills D. R., Kramer F. R. Localization of the Q beta replicase recognition site in MDV-1 RNA. J Biochem. 1983 Mar;93(3):669–674. doi: 10.1093/jb/93.3.669. [DOI] [PubMed] [Google Scholar]
  24. Novak J. E., Kirkegaard K. Coupling between genome translation and replication in an RNA virus. Genes Dev. 1994 Jul 15;8(14):1726–1737. doi: 10.1101/gad.8.14.1726. [DOI] [PubMed] [Google Scholar]
  25. Restrepo-Hartwig M. A., Ahlquist P. Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis. J Virol. 1996 Dec;70(12):8908–8916. doi: 10.1128/jvi.70.12.8908-8916.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ross J. mRNA stability in mammalian cells. Microbiol Rev. 1995 Sep;59(3):423–450. doi: 10.1128/mr.59.3.423-450.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schuppli D., Miranda G., Qiu S., Weber H. A branched stem-loop structure in the M-site of bacteriophage Qbeta RNA is important for template recognition by Qbeta replicase holoenzyme. J Mol Biol. 1998 Oct 30;283(3):585–593. doi: 10.1006/jmbi.1998.2123. [DOI] [PubMed] [Google Scholar]
  28. Schuppli D., Miranda G., Tsui H. C., Winkler M. E., Sogo J. M., Weber H. Altered 3'-terminal RNA structure in phage Qbeta adapted to host factor-less Escherichia coli. Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10239–10242. doi: 10.1073/pnas.94.19.10239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Siegel R. W., Bellon L., Beigelman L., Kao C. C. Moieties in an RNA promoter specifically recognized by a viral RNA-dependent RNA polymerase. Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11613–11618. doi: 10.1073/pnas.95.20.11613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Singh R. N., Dreher T. W. 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. RNA. 1998 Sep;4(9):1083–1095. doi: 10.1017/s1355838298980694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Song C., Simon A. E. RNA-dependent RNA polymerase from plants infected with turnip crinkle virus can transcribe (+)- and (-)-strands of virus-associated RNAs. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8792–8796. doi: 10.1073/pnas.91.19.8792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Song C., Simon A. E. Requirement of a 3'-terminal stem-loop in in vitro transcription by an RNA-dependent RNA polymerase. J Mol Biol. 1995 Nov 17;254(1):6–14. doi: 10.1006/jmbi.1995.0594. [DOI] [PubMed] [Google Scholar]
  33. Stupina V., Simon A. E. Analysis in vivo of turnip crinkle virus satellite RNA C variants with mutations in the 3'-terminal minus-strand promoter. Virology. 1997 Nov 24;238(2):470–477. doi: 10.1006/viro.1997.8850. [DOI] [PubMed] [Google Scholar]
  34. Weiland J. J., Dreher T. W. Cis-preferential replication of the turnip yellow mosaic virus RNA genome. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6095–6099. doi: 10.1073/pnas.90.13.6095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zamora H., Luce R., Biebricher C. K. Design of artificial short-chained RNA species that are replicated by Q beta replicase. Biochemistry. 1995 Jan 31;34(4):1261–1266. doi: 10.1021/bi00004a020. [DOI] [PubMed] [Google Scholar]
  36. van Rossum C. M., Garcia M. L., Bol J. F. Accumulation of alfalfa mosaic virus RNAs 1 and 2 requires the encoded proteins in cis. J Virol. 1996 Aug;70(8):5100–5105. doi: 10.1128/jvi.70.8.5100-5105.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES