3a 3b start site determined in this experiment contained an RNA cap structure identical with that present at the 5' end of nearly all eukaryotic mRNAs (see Figure 4-13). This 5' cap was added by enzymes in the nuclear extract, which can add a cap only to an RNA that has a 5' tri- or diphosphate. Because a 5' end generated by cleavage of a longer RNA would have a 5' monophosphate, it could not be capped. Consequently, researchers concluded that the capped nucleotide generated in the in vitro transcription reaction must have been the nucleotide with which transcription was initiated. The finding that the sequence at the 5' end of the RNA transcripts produced in vitro is the same as that at the 5' end of late adenovirus mRNAs isolated from cells confirmed that the capped nucleotide of adenovirus late mRNAs coincides with the transcription-initiation site.
Similar in vitro transcription assays with other cloned eukaryotic genes have produced similar results. In each case, the start site was found to be equivalent to the capped 5' sequence of the corresponding mRNA. Thus synthesis of eukaryotic precursors of mRNAs by RNA polymerase II begins at the DNA sequence encoding the capped 5' end of the mRNA. Today, the transcription start site for a newly characterized mRNA generally is determined simply by identifying the DNA sequence encoding the 5' end of the mRNA.
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