Nucleic Acids Research

We show that in E. coli, a Chlamydomonas chloroplast promoter, PA, is repressed by Integration Host Factor (IHF). The himA42 mutation, altering the a-subunit of E. coli IHF, leads to over-accunulation of PA transcripts in vivo. This effect requires upstream chloroplast DNA sequences. DNAase I and methylation protection experiments show that IHF binds in vitro to a site within PA and band-retardation shows that IHF inhibits formation of PA-E. coli RNA polymerase open complexes. We interpret these results, together with our previous deletion analyses, to mean that in E. coli, repression of PA by IHF minimally requires both binding of IHF to a site overlapping PA and binding of one or more additional proteins, perhaps including IHF itself, to sequences upstream of PA. INTRODUCTION Interaction of RNA polymerase with promoter regions and clearing from these regions can be modulated by DNA structure and topology and by repressors or activators whose target sites overlap. Proteins binding to DNA at a distance may also affect initiation of transcription. It is now becoming apparent that all these regulatory mechanisms operate in both prokaryotes and eukaryotes (for reviews see refs. 1-3). Chloroplast promoters share several features with bacterial promoters: similar consensus sequences in the so-called "-35 region" and in the "-10 region", separated by approximately 17 bp. The similarity of chloroplast and E. coli promoters is thought to reflect their function, since at least some chloroplast promoters are recognized by E. coli RNA polymerase in vivo and in vitro (for reviews see refs. 4 and 5). Little is known, however, about proteins that modulate chloroplast transcription. We are searching for such regulatory proteins by analyzing the regulation of a Chlamydomonas chloroplast promoter that we call PA. We have shown that regulation of PA in E. coli involves upstream chloroplast DNA sequences, suggesting that PA might be regulated by trans-acting transcriptional factors acting at a © I R L Press Limited, Oxford, England. 3313 Nucleic Acids Research distance. We also noted the presence of several chloroplast DNA sequences resembling the consensus E. coli IHF binding sequence, one of these overlapping PA (6). IHF is a member of a family of small, basic, DNA binding proteins which are ubiquitous in prokaryotes (7,8) . In E. coli, the protein is a heterodimer consisting of one a-subunit and one S-subunit (9). It specifically binds to the consensus sequence YAA.RTTGAT(A,T) (10,11) and affects phage X site-specific recombination (12), specificity of transposition of IS1 (13), inversion of a DNA segment that controls phase variation in E. coli (14), replication of certain plasmids (15,16), transcription from certain promoters (17-22), and possibly translation of certain mRNAs (23). Since IHF appears to be one factor which mediates the response of certain promoters to supercoiling (N.P. Higgins, personal communication), and since supercoiling modulates PA both in E. coli and in chloroplasts (6), we tested a possible role of IHF in regulating PA using three approaches: (1 ) by determining the effects of a himA mutation altering the ar-subunit of IHF (24) on PA activity in vivo using a primer extension assay; (2) by protecting specific sequences in the PA region in vitro with IHF from nuclease digestion and methylation; and (3) by determining the effects of IHF on open complex formation between RNA polymerase and PA in vitro using a gel-retardation assay. Together, our results indicate that IHF represses PA by binding to PA and inhibiting open complex formation. MATERIALS AND METHODS Bacteria E. coli strain UT481 (met-, thy-, A(pro lac), r-m-, supD, F'::TnlO, traD36, proAB, lacIq, lacZ, AM15) was from C. Lark and strains N99 (Strr, sup°, galK-, F-) and HN414 (Strr, sup°, galK-, F-, himA42) were from N.P. Higgins. Growth conditions and isolation of RNA for primer extension experiments have been described (6). P lasmids Construction of plasmids pRT11, pRTlldel4, pRTlldel3O, pRTlldel32, and pRT11del3 has been described (6). Plasmids pRT11 and pRT11del3O were used to transform E. coli strains N99 and HN414 by standard techniques (25). Primers The T7 promoter primer was from Promega Biotec. Primer #62 and the 5' end labeling of this primer have been described (6).