THE NUCLEIC ACID-BINDING ZINC-FINGER PROTEIN OF POTATO-VIRUS-M IS TRANSLATED BY INTERNAL INITIATION AS WELL AS BY RIBOSOMAL FRAMESHIFTING INVOLVING A SHIFTY STOP CODON AND A NOVEL MECHANISM OF P-SITE SLIPPAGE

The genes for the capsid protein CP and the nucleic acid-binding 12K p rotein (pr12) of potato virus M (PVM) constitute the 3' terminal gene cluster of the PVM RNA genome. Both proteins are presumably translated from a single subgenomic RNA. We have identified two translational st rategies operating in pr12 gene expression. Internal initiation at the first and the second AUG codon of the pr12 coding sequence results in the synthesis of the 12K protein. In addition the protein is produced as a CP/12K transframe protein by ribosomal frameshifting. For these studies parts of the CP and pr12 coding sequences including the putati ve frameshift region were introduced into an internal position of the beta-glucuronidase gene. Mutational analyses in conjunction with in vi tro translation experiments identified a homopolymeric string of four adenosine nucleotides which together with a 3' flanking UGA stop codon were required for efficient frameshifting. The signal AAAAUGA is the first frameshift signal with a shifty stop codon to be analyzed in the eukaryotic system. Substitution of the four consecutive adenosine nuc leotides by UUUU increased the efficiency of frameshifting, while subs titution by GGGG or CCCC dramatically reduced the synthesis of the tra nsframe protein. Also, UAA and UAG could replace the opal stop codon w ithout effect on the frameshifting event, but mutation of UGA to the s ense codon UGG inhibited transframe protein formation. These findings suggest that the mechanism of ribosomal frameshifting at the PVM signa l is different from the one described by the 'simultaneous slippage' m odel in that only the string of four adenosine nucleotides represents the slippery sequence involved in a -1 P-site slippage.