In vitro experiments with model promoters first showed that RNA polymerase II molecules that initiate transcription have an unphosphorylated CTD. Once the polymerase initiates transcription and begins to move away from the promoter, many of the serine and some tyrosine residues in the CTD are phosphorylated. Analysis of polytene chromosomes from Drosophila salivary glands prepared just before molting of the larva indicate that the CTD also is phosphorylated during in vivo transcription. The large chromosomal "puffs" induced at this time in development are regions where the genome is very actively transcribed. Staining with antibodies specific for the phosphorylated or unphosphorylated CTD demonstrated that RNA polymerase II associated with the highly transcribed puffed regions contains a phosphory-lated CTD (Figure 11-7).
RNA Polymerase II Initiates Transcription at DNA Sequences Corresponding to the 5' Cap of mRNAs
Several experimental approaches have been used to identify DNA sequences at which RNA polymerase II initiates transcription. Approximate mapping of the transcription start
▲ EXPERIMENTAL FIGURE 11-8 In vitro transcription of restriction fragments and measurement of the RNA lengths localize the initiation site of the adenovirus major late transcription unit. (a) The top line shows restriction sites for Hindlll (black), Xmalll (blue), and Smal (red) in the region of the adenovirus genome where the transcription-initiation site was located by nascent-transcript analysis (near 16 map units). The Hindlll, Xmalll, and Smal restriction fragments that encompass the initiation site were individually incubated with a nuclear extract prepared from cultured cells and 32P-labeled ribonucleoside triphosphates. Transcription of each fragment begins at the start site and ends when an RNA polymerase ll molecule "runs off" the cut end of the fragment template, producing a run-off transcript (wavy red lines). (b) The run-off transcripts synthesized site is possible by exposing cultured cells or isolated nuclei to 32P-labeled ribonucleotides for very brief times, as described earlier. After the resulting labeled nascent transcripts are separated on the basis of chain length, each size fraction is incubated under hybridization conditions with overlapping restriction fragments that encompass the DNA region of interest. The restriction fragment that hybridizes to the shortest labeled nascent chain, as well as all the longer ones, contains the transcription start site. One of the first transcription units to be analyzed in this way was the major late transcription unit of adenovirus. This analysis indicated that transcription was initiated in a region ~6 kb from the left end of the viral genome.
The precise base pair where RNA polymerase II initiates transcription in the adenovirus late transcription unit was determined by analyzing the RNAs synthesized during in vitro transcription of adenovirus DNA restriction fragments that extended somewhat upstream and downstream of the approximate initiation region determined by nascent-transcript analysis. The rationale of this experiment and typical results are illustrated in Figure 11-8. The RNA transcripts synthesized in vitro by RNA polymerase II from the from each fragment were then subjected to gel electrophoresis and autoradiography to determine their exact lengths. Since the positions of the restriction sites in the adenovirus DNA were known, the lengths of the run-off transcripts in nucleotides (nt) produced from the restriction fragments precisely map the initiation site on the adenovirus genome, as diagrammed in part (a). In the gels shown here, the bands at the top and bottom represent high- and low-molecular-weight RNA transcripts that are formed under the conditions of the experiment. The sample in lane 3b is the same as that in lane 3a, except that a-amanitin, an inhibitor of RNA polymerase II, was included in the transcription mixture. See the text for further discussion. [See R. M. Evans and E. Ziff, 1978, Cell 15:1463, and P A. Weil et al., 1979, Cell 18:469. Autoradiogram courtesy of R. G. Roeder.]
HindIII XmaIII Sma
HindIII XmaIII SmaI a-amanitin
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