In vitro transcription experiments using purified RNA polymerase II, a protein extract prepared from the nuclei of cultured cells, and DNA templates containing sequences encoding the 5′ ends of mRNAs for a number of abundantly expressed genes revealed that the transcripts produced always contained a cap structure at their 5′ ends identical to that present at the 5′ end of the spliced mRNA normally expressed from the gene in vivo (see Figure 5-14). In these experiments, the 5′ cap was added to the 5′ end of the nascent RNA 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 would not be capped. Consequently, researchers concluded that the capped nucleotides generated in the in vitro transcription reactions must have been the nucleotides with which transcription was initiated. Sequence analysis revealed that, for any given gene, the sequence at the 5′ end of the RNA transcripts produced in vitro is the same as that at the 5′ end of the mRNAs isolated from cells, confirming that the capped nucleotide of eukaryotic mRNAs coincides with the transcription start site. Today the transcription start site for a newly characterized mRNA is generally determined simply by identifying the DNA sequence encoding the 5′-capped nucleotide of the encoded mRNA.