Control of TGEV mRNA Transcription
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Coronavirus proteins are translated from a nested set of subgenomic mRNAs which have common 3’ termini with unique 5’ extensions. Evidence suggests that coronavirus mRNAs are generated by a mechanism of leader primed transcription. Leader RNA binds to consensus sequences upstream of each gene on full length negative strand viral RNA and transcription proceeds to the 5’ end of the negative strand to produce the nested set of mRNAs. Even though this gives rise to polycistronic mRNA species only the 5’ extension of each mRNA is translated to give the viral proteins. The leader RNA for TGEV is about 90 nucleotides long and contains the sequence which recognises the leader binding sites on the negative strand RNA. Evidence suggests that the length of the leader binding sequence may be involved in transcriptional control of individual mRNAs. In order to investigate this a virus specific mRNA isolation method was developed to measure the relative amounts of mRNAs synthesised during an infection of LLC-PK1 cells with TGEV (strain FS772/70). Thus the relative quantity of each mRNA can be determined and correlated with the variation in size of the leader binding site.
KeywordsMagnetic Bead Negative Strand Guanidinium Isothiocyanate Streptavidin Magnetic Bead Incomplete Transcript
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