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      cis acting RNA sequences control the gag-pol translation readthrough in murine leukemia virus

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          Abstract

          The pol gene of the Moloney murine leukemia virus (M-MuLV) is expressed as a Gag-Pol fusion protein through an in-frame suppression of the UAG termination codon located between the two genes. The role of nucleotide context in suppression was investigated, in a rabbit reticulocyte lysate translation system, using site-directed mutagenesis. The results indicate that the translational readthrough is mediated by at least 50 bases long RNA sequence located 3′ to the gag UAG termination codon. Within this sequence a short purine-rich sequence adjacent to the amber codon, highly conserved among different retroviruses, appears essential for M-MuLV suppression. Two alternative putative stem and loop like RNA structures can be drawn at the gag-pol junction, one abutting the gag UAG codon, and the second downstream to it. None of these structures appears to be important to the suppression process.

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          Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.

          D Melton, P. Krieg, M Rebagliati … (1984)
          A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).
          Article 0 comments Cited 191 times – based on 0 reviews     Review now
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            Signals for ribosomal frameshifting in the rous sarcoma virus gag-pol region

            Tyler Jacks, Hiten Madhani, Frank R. Masiarz … (1988)
            The gag-pol protein of Rous sarcoma virus (RSV), the precursor to the enzymes responsible for reverse transcription and integration, is expressed from two genes that lie in different translational reading frames by ribosomal frameshifting. Here, we localize the site of frameshifting and show that the frameshifting reaction is mediated by slippage of two adjacent tRNAs by a single nucleotide in the 5′ direction. The gag terminator, which immediately follows the frameshift site, is not required for frameshifting. Other suspected retroviral frameshift sites mediate frameshifting when placed at the end of RSV gag. Mutations in RSV pol also affect synthesis of the gag-pol protein in vitro. The effects of these mutations best correlate with the potential to form an RNA stem-loop structure adjacent to the frameshift site. A short sequence of RSV RNA, 147 nucleotides in length, containing the frameshift site and stem-loop structure, is sufficient to direct frameshifting in a novel genetic context.
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              Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting.

              H Varmus, T Jacks (1985)
              The pol gene of Rous sarcoma virus is positioned downstream of the gag gene in a different, briefly overlapping reading frame; nevertheless, the primary translation product of pol is a gag-pol fusion protein. Two mechanisms, ribosomal frameshifting and RNA splicing, have been considered to explain this phenomenon. The frameshifting model is supported by synthesis of both gag protein and gag-pol fusion protein in a cell-free mammalian translation system programmed by a single RNA species that was synthesized from cloned viral DNA with a bacteriophage RNA polymerase. Under these conditions, the ratio of the gag protein to the fusion protein (about 20 to 1) is similar to that previously observed in infected cells, the frameshifting is specific for the gag-pol junction, and it is unaffected by large deletions in gag. In addition, synthesis of the fusion protein is ten times less efficient in an Escherichia coli cell-free translation system and cannot be explained by transcriptional errors or in vitro modification of the RNA. Ribosomal frameshifting may affect production of other proteins in higher eukaryotes, including proteins encoded by several retroviruses and transposable elements.
              Article 0 comments Cited 74 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Virology
                Journal ID (iso-abbrev): Virology
                Title: Virology
                Publisher: Published by Elsevier Inc.
                ISSN (Print): 0042-6822
                ISSN (Electronic): 1096-0341
                Publication date PMC-release: 22 July 2004
                Publication date (Print): July 1991
                Publication date (Electronic): 22 July 2004
                Volume: 183
                Issue: 1
                Pages: 313-319
                Affiliations
                [a ]Departments of Molecular Genetics and Virology The Hebrew University, Hadassah Medical School, Jerusalem, 91010, Israel
                [∗ ]Department of Internal Medicine A, The Hebrew University, Hadassah Medical School, Jerusalem, 91010, Israel
                Author notes
                [1 ]To whom reprint requests should be addressed.
                Article
                Publisher ID: 0042-6822(91)90144-Z
                DOI: 10.1016/0042-6822(91)90144-Z
                PMC ID: 7131665
                PubMed ID: 2053284
                SO-VID: 18b4dc10-e086-4be4-ab7e-7dff3f72accb
                Copyright statement: Copyright © 1991 Published by Elsevier Inc.
                License:

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                Date received : 10 January 1991
                Date accepted : 28 March 1991
                Categories
                Subject: Article

                ScienceOpen disciplines: Microbiology & Virology
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                ScienceOpen disciplines: Microbiology & Virology

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