A 3,466-bp nucleotide sequence containing the katE gene of Escherichia coli has been determined. An open reading frame of 2,259 bp was found and was preceded by a potential ribosome-binding site. The predicted N-terminal sequence agreed with the sequence determined by direct amino acid sequencing, and the predicted direction of transcription was confirmed by expression of the gene cloned in both directions behind a T7 promoter. The start site of transcription was determined to be 127 bp upstream from the start of the open reading frame, and a potential RNA polymerase-binding site similar to a sequence preceding the xthA gene, which is also controlled by the KatF protein, was identified. The predicted sequence of the 753-amino-acid protein was comnpared with known sequences of other catalases, revealing significant similarity to the shorter catalases, including the residues in the putative active site and residues involved in heme binding.Escherichia coli produces two catalases, a bifunctional catalase-peroxidase (hydroperoxidase I [HPI]) and a monofunctional catalase (HPII). Both enzymes have been purified and characterized to be quite different from each other and from the typical catalase, which is active as a tetramer of 65,000-Da subunits and four protoheme IX groups. HPI was found to be a tetramer of 78,000-Da subunits with just two protoheme IX groups per tetramer and with an associated broad-spectrum peroxidase activity (6). HPII was purified (7) and characterized as a hexamer of 93,000-Da subunits (18) and six heme d-like groups (5) per hexamer. The genes encoding HPI and HPII, katG and katE, respectively, are unlinked, mapping at 89.2 (21) and 37.2 (17) min, respectively. A third gene, katF, mapping at 59.0 min (20), was found to be required for expression of katE but not katG. The katF gene has been cloned (28) and sequenced (27), revealing a striking similarity between the KatF protein and known sigma transcription factors, suggesting its mechanism as a positive effector of katE (27), of xthA (32), and possibly of other genes involved in resistance to near-UV radiation (33).The katG gene has been cloned (22, 42) and sequenced (41) and predicts an amino acid sequence that bears no resemblance to any of the known catalase sequences. Instead, a striking resemblance to a peroxidase from Bacillus stearothermophilus has been observed (23), suggesting that the bifunctional HPI is more closely related to the family of peroxidases than catalases. On the basis of its very different physical structure, it seemed unlikely that HPII would have any sequence or structural similarity to the common catalases. This paper describes the sequence analysis of katE, the identification of the site of transcription initiation, and a comparison of the predicted amino acid sequence of HPII with the sequences of other catalases. MATERIALS AND METHODSBacterial strains and plasmids. The strains JM101 (45) and NM522 (25) were used for plasmid preparations and for the preparation of single-stranded DNA for sequencing. Cul-* Correspo...

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is important in maintaining the extracellular proteolytic balance during tissue remodeling processes. To allow homeostatic tissue turnover, the murine Timp-1 gene is expressed by most cells at a low basal level, and during acute remodeling its transcription is activated by a variety of stimuli. A non-consensus AP-1-binding site (5-TGAGTAA-3) that is conserved in mammalian timp-1 genes is a critical element in basal and serum-stimulated transcription. We show here that each strand of this unusual AP-1 site binds a distinct single-stranded DNA-binding protein, although neither strand from a perfect consensus AP-1 site from the human collagenase gene shows similar binding. One of the single-strand binding factors, which we term ssT1, binds to a second upstream Timp-1 region between nucleotides ؊115 and ؊100. Deletion analysis demonstrated that this region is important in basal but not serum-inducible transcription. The ssT1 factor was 52-54 kDa by UV cross-linking of electrophoretic mobility shift assays and Southwestern blot analysis. Its binding to DNA shows sequence selectivity rather than specificity, with 5-CT/ATTN (4 -6)ATC-3 as a favored motif. Multiple ssT1-like activities were found in nuclear extracts from mouse fibroblasts and rat liver and testis, suggesting that these factors may regulate basal Timp-1 transcription in a tissue-specific fashion.Extracellular matrix turnover is highly regulated at multiple levels, a critical one being the balance between the levels of matrix metalloproteinases (MMPs) 1 and their specific inhibitors, the TIMPs (tissue inhibitors of metalloproteinases (1, 2)).

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