Abstract
Bacteriophage T4 late promoters display a conserved sequence that extends over about 18 base pairs, in which the central 8-base pair sequence (TATAAATA in the nontranscribed strand) is absolutely conserved. Transcription by T4-modified RNA polymerase in vitro nevertheless initiates within a cluster of 6 overlapping variant T4 late promoters that deviate from the absolutely conserved sequence at one or two positions. One of these variant promoters is dominant, and its sequence implies that two of the absolutely conserved nucleotides (underlined above) are not essential. Three other variant promoters that contain only one of the deviations found in the dominant variant promoter are, at best, utilized weakly, suggesting that sequences outside the absolutely conserved segment are important for promoter function. A newly devised method, based on arrest of RNA chain elongation with ribonucleotide analogs and its reversal, has been used to precisely map initiation within the overlapping promoter cluster. Multiple cycles of RNA chain arrest, pyrophosphorolysis, and resynthesis can be executed. This process permits a precise stepwise control of the advance of a transcription complex through a gene.
Highlights
A newly devised method, based on arrest of RNA Aparftromthe absolutelcyonserved -10 sequence, chain elongation with ribonucleotide analogs and its TATAAATA, a weaker homology among the 14 known T4 reversal, has been used to precisely map initiation late promotersequences extends to the start soiftetranscripwithin the overlapping promoter cluster
Yeast tRNA was from GIBCO, and E. coli 5 S RNA was from nents were added to preformed transcription complexes in the above
Lock-step Transcription-The ability to reverse the RNA counting the bands from lunes g-i, it was possible to quantichain termination by 3’0MeNTPs suggested the following tatively assign relative initiation rates; 82%of chains started scheme for mapping both the major and the minor sites of at position 22 in the sequence shown in Fig. 2 and in Fig. 7, initiation in the variant promoter region (Fig.7)
Summary
A newly devised method, based on arrest of RNA Aparftromthe absolutelcyonserved -10 sequence, chain elongation with ribonucleotide analogs and its TATAAATA, a weaker homology among the 14 known T4 reversal, has been used to precisely map initiation late promotersequences extends to the start soiftetranscripwithin the overlapping promoter cluster. When the plasmid containing this region (pTF1) was used as templatefor transcription by late T4-modified RNA polymerase, a 380-nucleotide RNA product was made (datanot shown, but see Fig. 3)indicating that at least one of the variant promoter sequences was being utilized.
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