INITIATION OF TRANSCRIPTION BY BACTERIOPHAGE T3 RNA POLYMERASE (MOLECULAR BIOLOGY, T-PHAGES, TRANSCRIPTION FACTORS)
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The infection of Escherichia coli with bacteriophage T3 leads to the induction of a new DNA-dependent RNA polymerase of M(,r) = 100,000. It is encoded by gene 1 of the phage and is expressed along with other class I genes by the E. coli RNA polymerase. This enzyme carries out the transcription of all "late" (class II and class III) genes. It shows a remarkable specificity towards T3 DNA and carries out specific initiation, elongation and termination of RNA synthesis without the use of any other protein factors.;In order to study the regulatory signals recognized by the T3 RNA polymerase, the major "start sites" of transcription were located relative to a restriction map of T3 DNA. The DNA sequences around several such sites located in the class III region (45-100 map units) were determined and the exact start site ascertained by sequencing the transcript from the 5' end. A 23-base pair consensus "promoter".;sequence for T3 RNA polymerase was derived on the basis of this determination. When this sequence, (UNFORMATTED TABLE FOLLOWS).;-10 +1.;A A T T A A C C C T C A C T A A A G G G A G A,;(TABLE ENDS).;was compared with those of the related bacteriophages T7 and SP6, the overall sequence was found be remarkably similar, with the only consistent difference around the -10 region. On the basis of these studies, the "-10 region" was designated the "specificity element" recognized by these bacteriophage-encoded RNA polymerases which show little recognition of the heterologous phase promoters.;The interaction of T3 RNA polymerase with its cognate promoter was studied by "footprinting" techniques. These studies revealed an absolute requirement for the initiating nucleotide, GTP, for the phage RNA polymerase to specifically bind to and protect from DNase I digestion, the T3 promoter. In the absence of the initiating nucleotide the enzyme binds DNA at random with less affinity. Nitrocellulose binding studies and studies on the interaction of T7 RNA polymerase with its cognate promoter revealed a similar requirement for the initiating nucleotide, GTP. On the basis on these observations, it is proposed that the binding of the initiating nucleotide (in this case, GTP) drives the T3 RNA polymerase into a promoter-specific "initiation conformation".
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