Abstract
We previously reported (Liang, Q., He, J.-S., and Fulco, A.J. (1995) J. Biol. Chem. 270, 4438-4450) that Bm3R1, a repressor regulating the expression of P450BM-3 in Bacillus megaterium, could bind to Barbie box sequences in the 5'-flanking regions of barbiturate-inducible genes. We've now shown that pentobarbital does not inhibit in vitro binding of Bm3R1 to the P450BM-3 and P450BM-1 Barbie boxes (BB3 and BB1), although the palindromic operator sequence (OIII) of P450BM-3 did have a strong competitive effect on such binding. G39E-Bm3R1, a mutant of Bm3R1, did not bind to either Barbie box. In the presence of Bm3R1, portions of the regulatory regions of P450BM-3 and P450BM-1 were protected from DNase I digestion. These included 11 of the 15 base pairs of BB3 plus 7 base pairs 3' to BB3, BB1 plus 16 base pairs 3' to BB1, and, in the 5'-flanking region of P450BM-1, segments covering most of two palindromic sequences (OII and OIII) of 24 and 52 base pairs. These DNase I-protected regions (including OIII) showed considerable sequence identity, especially in a conserved poly(A) motif. Barbiturates did not inhibit binding of Bm3R1 to OI. OII in vitro while G39E-Bm3R1 did not bind. The regulatory effects of Bm3R1 on P450BM-1 and P450BM-3 were also evaluated in vivo using heterologous chloramphenicol acetyltransferase constructs and Western blotting. In the G39E mutant strain, both P450BM-1 and P450BM-3 were constitutively expressed, and the regulatory proteins Bm1P1 and Bm3P1, although still pentobarbital-inducible, had significantly higher basal levels of synthesis. In toto, our results show that Bm3R1 represses both P450BM-1 and P450BM-3 expression and that it may effect this by coordinate binding to operator and Barbie box sequences to produce looping of the P450BM-1 and P450BM-3 regulatory regions through protein-protein interaction.
Highlights
From the Department of Biological Chemistry and the Laboratory of Structural Biology and Molecular Medicine, School of Medicine, University of California, Los Angeles, California 90024-1737
Our results show that Bm3Rl represses both P450B M _1 and P450B M _3 expression and that it may effect this by coordinate binding to operator and Barbie box sequences to produce looping of the P450B M _1 and P450B M _3 regulatory regions through protein-protein interaction
Comparison of Binding Affinity of Wild-type Bm3R1 and G39E-Bm3R1 to Barbie Box Sequences-In a previous report (6), we found that Bm3Rl, a repressor of the P450B M _3 gene (2), interacted with Barbie box elements in the 5'flanking regions ofP450 structural genes of B. megaterium
Summary
After cutting with EcoRI, an end-labeled DNA fragment of 177 bp (corresponding to bp -44 to -221 of the 5' -flanking region of the P450B M _1 structural gene) containing the P450B M _1 operator sites was obtained from low melting point agarose gel electrophoresis using the Geneclean kit. -429) containing both BB, and operator sites All of these three radioactive labeled DNA fragments were used in gel mobility shift assays. P450B M _3 was dige pro sted mote with r S reg ail ion i and ncluding labe ltehdewPi4th50[aB-M323PBJdaNrbTiPe using the Klenow fragment of DNA polymerase This 447-bp DNA fragment was excised by HindUI digestion to render it labeled at one end and purified by low melt agarose gel electrophoresis and the Geneclean kit. Techniques for transformation of B. megaterium protoplasts by plasmids and subsequent regeneration were based on Imanaka's procedure (16)
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