5'-Conformation of capped alfalfa mosaic virus ribonucleic acid may reflect its independence of the cap structure or of cap-binding protein for efficient translation

Abstract

Most eukaryotic mRNA are characterized by the presence of a 5''-terminal cap structure (m7GpppN), and removal of the cap or translation of capped mRNA in the presence of cap analogs (m7G) results in most cases in a significant decrease in the translational efficiency of the mRNA. One way of explaining the importance of the 5''-cap is that cap-binding proteins recognize the cap structure, destabilize the mRNA secondary structure and thus allow the 40S ribosomal subunit to bind to the mRNA. Evidently, the translational efficiency of alfalfa mosaic virus RNA 4 (AMV-4 RNA), a naturally capped RNA, is not affected significantly by cap analogs or by removal of the cap. In order to examine the potential relationship between the function of the cap structure and secondary structure at the 5''-mRNA terminus, partial enzymatic digestion of capped AMV-4 RNA with single strand specific and double strand specific nucleases was performed, and the experimental data were compared with computer-generated models of AMV-4 secondary structure. The in vitro translatability of AMV-4 was examined as a function of increasing K concentration, conditons that are likely to increase mRNA secondary structure. The nuclease-digestion results demonstrate that under native ionic conditions, the 5''-terminus of AMV-4 RNA is predominantly single stranded, although computer modeling and double-strand nuclease digestions indicate that the 5''-terminus can form weak base pairs with internal regions of the molecule. Binding of ribosomes to AMV-4 in vitro is relatively resistant to increased K concentration, again indicating that the 5''-terminus of AMV-4 RNA does not form stable base pairs. The data are consistent with a model wherein those mRNA with little potential for formation of stable base pairs in the 5''-terminal region preceding the initiation codon have a decreased dependence on the cap structure and cap-binding protein for efficient translation.