Nucleotide sequence of region preceding trp mRNA initiation site and its role in promoter and operator function.

Abstract

The nucleotide sequence of the region preceding the transcrption initiation site of the tophan oeron of Escherichia co was determined. Essentially all of the trp operator precedes the transcribed portion of the operon. The deduced sequence contains the recognition site of endonuclease Hpa I. This site is protected from Hpa I cleavage by RNA polymerase and by tip repressor. Regions of 2-fold symmetry are present in the DNA sequence.In most 080 and X-+80 trp transducing phages, the trp operon replaces a segment of the phage N operon and is transcribed with normal orientation by read-through transcription initiated at the phage N promoter (1, 2). We prepared 32P-labeled read-through RNA in vio and in vitro and employed selective hybridization to the denatured DNA of appropriate tip transducing phages to isolate the segment of these transcripts that immediately precedes the 5' end of the normal trp operon messenger RNA.Here we report the sequence of 33 nucleotides preceding the transcription initiation site of the tip operon. We show that the corresponding segment of DNA contains sequences essential for promoter and operator function. MATERIALS AND METHODSPreparation of RNA. The read-through RNA transcript containing the sequence preceding the 5' end of tip mRNA was labeled either in vitro using &80tip DNA as a template or in vivo by infecting cells with 080tip phages (Fig. 1).In Vitro Synthesis. Synthesis was performed in a coupled transcription-translation system essentially as described (3, 4).As templates, DNAs of phages 4080trpALD102, 480trp-ALC145-2, and q80trpALC1415 were used (Fig. 1). a-32P-labeled nucleoside triphosphates (used at a concentration of 50 ,MM) were purchased from New England Nuclear Corp. (specific activity Ci/mmol) through the kind efforts of Dr.W. Salser. Incubations were for 60 min at 340. RNA was isolated (5) and sequences corresponding to the promoter-operator region were trapped by two consecutive liquid hybridizations to complementary separated DNA strands at 510 for 16-18 hr (6) and at 370 in 30% formamide (5) 00. The products were separated as described for a normal fingerprint except that electrophoresis was performed for 35 min at 5000 V and homochromatography mix "C-2" (9) was used for development in the second dimension. RESULTSCharacterization of RNA complementary to the promoter-operator region of the tip operon DNAs from several c80trp transducing phages were used as templates for the in vitro synthesis of 32P-labeled RNA in a coupled transcription-translation system (4). Promoter-operator RNA, purified as described in Materials and Methods, is defined at the 5'-terminus by the phage-bacterial fusion point of the template DNA, 080trpALD102, and at the 3' end by the end point of the LD102 deletion. This RNA, a fingerprint of which is shown ( Fig. 2A), exhibited the mobility expected for a molecule of 150 4 10 nucleotides when electrophoresed on a denaturing polyacrylamide gel. A RNase T1 fingerprint of the 150 nucleotide band was identical to that in Fig. 2A. The fin...

RNA polymerase binding sites in λp lac 5 DNA

“…4, all of the fragments except the 1200-basepair fragment, which comes from the region immediately to the right of PL in Fig. 2 (fragment 7), are lost from the nitro-P/D 11 Fig. 5, that at least one more site on Xplac5 DNA can be protected under our conditions.…”

Transcription termination at the trp operon attenuators of Escherichia coli and Salmonella typhimurium: RNA secondary structure and regulation of termination.

“…These bands (Fig. 4 right) include the regions from approximately residues 42 to 74 (band 2) and residues 96 to 125 (band 3). Band 3 appears to be comparable in location to band 2 from E. coli.…”

Escherichia coli tryptophan repressor binds multiple sites within the aroH and trp operators.

“…Binding of the corepressor, L-tryptophan increases the affinity of Trp repressor for operator DNA, leading to the formation of the ternary repressor DNA complex (Rose et al 1973). The binding of Trp repressor to its operators inhibits the initiation of transcription of the trpR, aroH, and trp operon messages by preventing RNA polymerase from binding to the promoters (Oppenheim et al 1980).The physical location of the trp operator was previously defined by deletion studies (Bennett et al 1976) and by the isolation of constitutive mutations within the trp operator {Bennett and Yanofsky 1978). These operator constitutive mutations map at positions -16, -15, -7, and -6 in the trp promoter sequence.…”