5'-Terminal 7-methylguanosine and mRNA function. The effect of enzymatic decapping and of cap analogs on translation of tobacco-mosaic-virus RNA and globin mRNA in vitro.
1. Decapped tobacco mosaic virus (TMV) RNA and rabbit globin mRNA were prepared by enzymic treatment of RNAs with nucleotide pyrophosphatase purified from potato. The extent of removal of 5'-terminal 7-methylguanosine 5'-monophosphate (m7GMP) from TMV RNA was at least 97% as estimated by labeling of the 5' termini in vitro with S-adenosyl[methyl-3H]methionine catalysed by vaccinia virus methyltransferases. 2. The effect of enzymic decapping was compared with the effect of cap analogs on mRNAs translation in a nuclease-treated rabbit reticulocyte lysate and in a wheat germ extract. When translation was studied at low K+ concentration, little or no dependence on 5'-terminal 7-methylguanosine was found with either cell-free system. The importance of the 5'-terminal cap for the efficient translation of TMV RNA and globin mRNA increased as the concentration of K+ in a protein-synthesis system was raised. In a reticulocyte lysate analogs and enzymic decapping had a similar effect on translation. In a wheat germ extract, mRNA decapping resulted in a more pronounced decrease of mRNA activity, presumably due to the increased susceptibility of decapped mRNAs to the nucleases present in this protein synthesis system. 3. The requirement for a 5'-terminal cap was similar for the synthesis of 130,000-Mr and 165,000-Mr polypeptides coded by TMV RNA. This indicates that both proteins may be initiated at the common site close to the 5' terminus.
D000818:Animals; D005914:Globins / Q000096:biosynthesis*; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D011188:Potassium / Q000494:pharmacology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014027:Tobacco Mosaic Virus / Q000378:metabolism*
D012333:RNA, Messenger; D006151:Guanosine; C016578:7-methylguanosine; D005914:Globins; D011188:Potassium