Detection of bacterial gene expression elements on Tobacco mosaic virus RNA using cDNA analysis.
Tobacco mosaic virus (TMV) is a positive-stranded RNA virus that infects plants. Interestingly, the 5'-untranslated region (UTR) of the TMV RNA genome is recognized and translated by the ribosomes of Escherichia coli in a Shine-Dalgarno (SD) sequence-independent manner. This study aimed at investigation of the bacterial recognition modules that control gene expression within the TMV RNA genome. To this end, the 5'-end-complete cDNA of the TMV RNA and several 5'-end-truncated cDNA mutants, in which the movement protein-encoding gene and its downstream region were replaced with a DNA sequence encoding a green fluorescent protein, i.e., monomeric Umikinoko-Green (mUkG1), were constructed. Surprisingly, mUkG1 fluorescence was observed in E. coli transformants harboring the cloned cDNAs, although they were inserted into a vector lacking a promoter. Analysis of the 5'-end-truncated cDNA mutants and promoter prediction suggested that an E. coli-specific promoter might be located 2.1 kb upstream of the initiation codon for mUkG1. Furthermore, Western blotting analysis and conversion of the initiation codon ATG to AGT indicated that the translation of mUkG1 started from the correct initiation codon. These results imply that E. coli ribosomes correctly recognize the initiation codon on the mRNA, irrespective of the overly long 5'-UTR. To the best of our knowledge, this report is the first to reveal a recognizable bacterial module hidden within the TMV RNA genome through cDNA construction.