In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides

Abstract

A plasmid containing the bacteriophage SP6 promoter, designated pHSTO, permits in vitro transcription of RNAs devoid of vector-derived nucleotides. This vector has been characterized for relative transcriptional activity using constructs which alter the conserved nucleotides extending beyond the SP6 transcriptional initiation site. SP6 polymerase efficiently transcribes cDNA inserts which contain a guanosine (G) nucleotide at position +1 relative to the SP6 promoter; however, inserts with an adenosine (A) or pyrimidine at position +1 are not transcribed. Several cellular and viral cDNAs have been transcribed into translatable messenger RNA using this vector; however, SP6 polymerase will not transcribe the A-T rich untranslated leader from alfalfa mosaic virus RNA 4 efficiently unless the viral mRNA cap site is separated from the transcriptional initiation site by twelve base pairs of vector DNA. Chimeric messenger RNAs were created by linking the untranslated leader sequence of several viral mRNAs to the coding region of barley alpha-amylase, and the resultant mRNAs were translated in a wheat germ extract to determine relative translational efficiencies. The untranslated leader sequences of turnip yellow mosaic virus coat protein mRNA and black beetle virus RNA 2 did not increase translational efficiency, while the tobacco mosaic virus leader stimulated translation significantly. The results indicate that substitution of a cognate untranslated leader sequence with a leader derived from a highly efficient mRNA does not necessarily predict enhanced translational efficiency of the chimeric mRNA.