STRUCTURAL-STUDIES AND FUNCTIONAL-STUDIES OF RETROVIRAL RNA PSEUDOKNOTS INVOLVED IN RIBOSOMAL FRAMESHIFTING - NUCLEOTIDES AT THE JUNCTION OF THE 2 STEMS ARE IMPORTANT FOR EFFICIENT RIBOSOMAL FRAMESHIFTING

Ribosomal frameshifting, a translational mechanism used during retrovi ral replication, involves a directed change in reading frame at a spec ific site at a defined frequency, Such programmed frameshifting at the mouse mammary tumor virus (MMTV) gag-pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream, Using in vitro translation assays and enzymati c and chemical probes for RNA structure, we have defined features of t he pseudoknot that promote efficient frameshifting. Heterologous RNA s tructures, e.g, a hairpin, a tRNA or a synthetic pseudoknot, substitut ed downstream of the shifty site fail to promote frameshifting, sugges ting that specific features of the MMTV pseudoknot are important for f unction. Site-directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide betw een the two stems, provides a specific structural determinant for effi cient frameshifting, Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) a lso promote frameshifting at the MMTV gag-pro shift site, dependent on the same structure at the junction of the two stems.