Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U(6)A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting

Lin, Zhaoru; Gilbert, Robert J. C.; Brierley, Ian

Assessment of in vitro frameshift efficiencies employed plasmids derived from pFScass 5 (12) . This vector contains the bacteriophage SP6 polymerase promoter driving expression of the influenza A/PR8/34 PB2 gene, with the minimal infectious bronchitis coronavirus (IBV) frameshifting signal (12) inserted at the Bgl II site at position 486 of the PB2 gene. We modified pFScass 5 by introducing a unique Mlu I site downstream of the inserted pseudoknot (creating plasmid pFScass 9), removed the entire IBV frameshifting signal using Bgl II and Mlu I and replaced it with a pair of complementary oligonucleotides encoding the AON-driven frameshifting signal utilized by Howard and colleagues (30) , giving plasmid pFSHIV-AON ( Figure 1 ). The frameshift signal in this plasmid comprises the HIV-1 slippery sequence U 6 A positioned 3 nt upstream of the binding site for a complementary AON. Frameshift assays in tissue culture cells employed derivatives of the dual luciferase reporter plasmid p2luc (33) . The AON frameshifting signal present in pFSHIV-AON was cloned as a pair of complementary oligonucleotides into Bam HI and Sal I-cut p2luc such that expression of the downstream firefly luciferase gene required either a À1 (p2lucAON À1) or a À2 (p2lucAON À2) frameshift at the end of the upstream Renilla luciferase gene. To allow calculation of frameshifting efficiencies, two control plasmids (p2lucOinc and p2lucOP1inc) were prepared in which the two luciferases were aligned in-frame in order to obtain readings for normalization of data. Expression of the U 6 A slippery sequence-derived À2 FS product in transfected cells employed plasmid pFSeGFP-N2. This plasmid was prepared by subcloning a polymerase chain reaction (PCR)-generated DNA fragment encoding the U 6 A slippery sequence, a 7 nt spacer and a stimulatory stemloop structure (see section 'Results') into XhoI/ BamHI-cut pEGFP-N2 (Clontech, GenBank Accession number U57608). In this plasmid, the eGFP tag is expressed only in the À2 frame (following a frameshift event) and the natural start codon of eGFP was changed (to TCG) by site-directed mutagenesis to minimize leaky expression of eGFP. All plasmid sequences were confirmed by dideoxy sequencing.