RNA stem loop structures influence the 3′ end formation of VLE-specific mRNAs <i>in vivo</i>.

(A) Schematic representation of recombinant pGKL1 elements where the G418R gene is followed by the coding sequence of wild-type (pRKL1-5) or modified (pRKL1-6, pRKL1-7) 3′ untranslated region of the K2ORF5 gene. TIR—terminal inverted repeat. (B) 3′ RACE-PCR analysis of individual mRNAs corresponding to the G418R gene expressed from modified pGKL1 elements. Samples were analyzed in 3.0% agarose gel stained by ethidium bromide. The strains used to purify the RNA are indicated above the lanes. M, DNA molecular mass marker (GeneRuler 100 bp Plus DNA Ladder, Fermentas). The respective values are indicated on the left. Specific products that were cloned to the pCR4-TOPO vector and used for sequencing are labelled with asterisks. Reverse transcription was carried out in the presence (+RT) and absence (-RT) of reverse transcriptase. (C) 3′ RACE-PCR analysis of the G418R gene from IFO1267_pRKL1-5 strain. In this and the following panels, the upper sequences on the right correspond to the template (plasmid) DNA; sequences situated below represent 3′ end regions of individual sequenced cDNA clones. Positions of the putative RNA stem loops are indicated above the sequences. Predicted RNA stem loops are displayed as cDNA nucleotide letters in circles on the left and the values of Gibbs free energy (ΔG) in kcal/mol are displayed for each structure. Stem loop distances from the gene stop codon are shown as numbers of nucleotides. The last few 3′ end nucleotides of the experimentally determined 3′ ends of cDNA are shown as letters enlarged proportionally to their occurrence (in %) in the sequenced clones in the case when these nucleotides were detected in at least two independent clones. (D) 3′ RACE-PCR analysis of the G418R gene from the IFO1267_pRKL1-6 strain. Mut, the mutated stem loop. (E) 3′ RACE-PCR analysis of the G418R gene from IFO1267_pRKL1-7 strain. Mut, the mutated stem loop; Res, the rescued stem loop.