Analysis of Cre1 binding sites in the Trichoderma reesei cbh1 upstream region

Abstract A 1.5-kb XbaI-SacII fragment containing the upstream region of the Trichoderma reesei cellobiohydrolase I gene ( cbh1 ) has been sequenced. The 1.5-kb fragment contains eight 6-bp sites having an identical or similar sequence to the consensus sequence for binding a catabolite repressor, Aspergillus nidulans CreA. Results of binding assays with the maltose-binding protein: :Cre1(10–131) fusion protein (Cre1 is a catabolite repressor of T. reesei ) and the cbhI upstream region revealed that a 504-bp XbaI-NspV fragment (nucleotide position − 1496 to − 993) bearing three 6-bp sites, Al, A2, and A3, and a 356-bp NspV-MunI fragment (nucleotide position −994 to −639) bearing three 6-bp sites, B1, B2, and B3, were shifted in the electrophoretic mobility shift assay. DNase I footprinting experiments showed that the 6-bp sites A2, B1, B2, and B3 were protected from DNase I digestion.     

Binding of the arginine repressor of Escherichia coli K12 to its operator sites.

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressors studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.     

Three additional operators, Op21, Op68, and Op88, of bacteriophage P1. Evidence for control of the P1 dam methylase by Op68

The repressor of bacteriophage P1, encoded by the c1 gene, is responsible for maintaining the P1 prophage in the lysogenic state. Previously, 11 c1 repressor binding sites or operators scattered over the whole genome of P1 have been found. From sequence analysis an asymmetric, 17-base pair consensus sequence, ATTGCTCTAATAAATTT, was derived. Using a synthetic 15-base-long oligodeoxyribonucleotide as operator probe, we have identified three additional operators. We have mapped the operators at the positions 21,68, and 88 of the P1 genome and determined their sequence. These operators are controlled by c1 because corresponding P1 DNA fragments (i) require c1 repressor in vivo in order to be clonable in multicopy plasmids, (ii) exhibit a c1-repressible promoter activity, (iii) are retarded by c1 repressor protein during electrophoresis, and (iv) contain the 17-base pair consensus sequence with one mismatch base each. Furthermore, we suggest that expression of the DNA adenine methylase (dam) encoded by P1 is controlled via Op68.     

ban operon of bacteriophage P1. Mutational analysis of the c1 repressor-controlled operator

The repressor of bacteriophage P1, encoded by the c1 gene, represses the phage lytic functions and is responsible for maintaining the P1 prophage in the lysogenic state. The c1 repressor interacts with at least 11 binding sites or operators widely scattered over the P1 genome. From these operators, a 17 base-pair asymmetric consensus sequence, ATTGCTCTAATAAATTT, was derived. Here, we show that the operator, Op72 of the P1ban operon consists of two overlapping 17 base-pair sequences a and b forming an incomplete palindrome. Op72a matches the consensus sequence, whereas Op72b contains two mismatches. The evidence is based on the sequence analysis of 27 operator mutants constitutive for ban expression. They were identified as single-base substitutions at positions 2 to 10 of Op72a (26 mutants) and at position 8 of Op72b (one mutant). We conclude from gel retardation and footprinting studies that two repressor molecules bind to the operator and that positions 4, 5 and 7 to 10 of the operator play an essential role in repressor recognition.     

N6-methyldeoxyadenosine residues at specific sites decrease the activity of the E1A promoter of adenovirus type 12 DNA

The activity of eukaryotic promoters is highly sensitive to site-specific modifications by DNA methylations. We have used the E1A promoter of adenovirus type 12 (Ad12) DNA to investigate the effects of methylations at different promoter sites on its activity. The chloramphenicol acetyltransferase gene has served as an activity indicator. Activity of the E1A promoter is lost or markedly decreased by deoxycytidine methylation of two HpaII (5'-C-C-G-G-3') or seven HhaI (5'-G-C-G-C-3') sites upstream from the 3' located T-A-T-A signal. There are two T-A-T-A signals in the E1A promoter of adenovirus type 12 DNA, one T-A-T-T-A-T sequence starting at nucleotide 276 (5' located), a second T-A-T-T-T-A-A sequence starting at nucleotide 414 (3' located). Deoxycytidine methylations at two AluI (5'-A-G-C-T-3') sites downstream from the 5' located T-A-T-A signal have no effect on promoter activity. When one EcoRI (5'-G-A-A-T-T-C-3') or one TaqI (5'-T-C-G-A-3') sequence at 281 base-pairs upstream or 61 base-pairs downstream from the 5' located E1A T-A-T-A signal, respectively, is deoxyadenosine methylated, the promoter becomes inactive. Deoxyadenosine methylation at one MboI (5'-G-A-T-C-3') site, which is located 127 nucleotides downstream from the 5' located T-A-T-A signal, fails to decrease E1A promoter activity. There is no conspicuous anatomical relation of any of these sites to the two presumptive enhancer sequences in the E1A promoter. We conclude that 5-deoxymethylcytidine or N6-methyldeoxyadenosine residues have to be introduced at highly specific promoter sites to inactivate the promoter. These sites are probably different for different promoters.     

DeltaG-based prediction and experimental confirmation of SYCRP1-binding sites on the Synechocystis genome

DNA-binding sites for SYCRP1, which is a regulatory protein of the cyanobacterium Synechocystissp. PCC6803, were predicted for the whole genome sequence by estimating changes in the binding free energy () for SYCRP1 for those sites. The values were calculated by summing DeltaDeltaG values derived from systematic single base-pair substitution experiments (symmetrical and cooperative binding model). Of the calculated binding sites, 23 sites with a value <3.9kcal.mol(-1) located upstream or between the ORFs were selected as putative binding sites for SYCRP1. In order to confirm whether SYCRP1 actually binds to these binding sites or not, 11 sites with the lowest values were tested experimentally, and we confirmed that SYCRP1 binds to ten of the 11 sites with a DeltaDeltaG(total) value <3.9kcal.mol(-1). The best correlation coefficient between and the observed DeltaDeltaG(total) for binding of SYCRP1 to those sites was 0.78. These results suggest that the DeltaDeltaG values derived from systematic single base-pair experiments may be used to screen for potential binding sites of a regulatory protein in the genome sequence.     

The c1 repressor of bacteriophage P1 operator-repressor interaction of wild-type and mutant repressor proteins.

The c1 repressor gene of bacteriophage P1 and the temperature-sensitive mutants P1c1.100 and P1c1.162 was cloned into an expression vector and the repressor proteins were overproduced. A rapid purification procedure was required for the isolation of the thermolabile repressor proteins. Identification of the highly purified protein of an apparent molecular weight of 33,000 as the product of the c1 gene was verified by (i) the coincidence of partial amino acid sequences determined experimentally to that deduced from the c1 DNA sequence, and (ii) the temperature-sensitive binding to the operator DNA of the thermolabile repressor proteins. Analysis of the products of c1-c1.100 recombinant DNAs relates the thermolability to an unknown alteration in the C-terminal half of the c1.100 repressor. Binding to the operator DNA of c1 repressor is sensitive to N-ethylmaleimide. Since the only three cysteine residues are located in the C-terminal half of the repressor it is suggested that this part of the molecule is important for the binding to the operator DNA. This assumption is supported by the findings that a 14-kDa C-terminal repressor fragment obtained by cyanogen bromide cleavage retains DNA binding properties.     

The c1 repressor inactivator protein coi of bacteriophage P1. Cloning and expression of coi and its interference with c1 repressor function

The immC region of bacteriophage P1 contains the c1 repressor gene and its upstream region with four c1-controlled operators and four open reading frames. A c1 inactivator gene, coi, was defined by mutations in immC that suppress the virulence of the P1virC mutation. The exact location of the coi gene was not known (Scott, J.R. (1980) Curr. Top. Microbiol. Immunol. 90, 49-65). When a variety of P1 immC fragments were inserted into an expression vector, a gene product was inducible for the open reading frame 4 only. We identify this product as the c1 inactivator protein, coi by the following criteria: (a) expression of coi from a recombinant plasmid induces the P1 prophage and inhibits lysogenization of sensitive bacteria by P1; (b) all c1-controlled operator-promoter elements tested in vivo are derepressed by coi; (c) a partially purified coi protein (apparent molecular weight = 4800) interacts with c1 repressor and inhibits its binding to the operator in vitro. Based on these results we refine a model for the regulation of those genes and elements within immC which participate in the decision of P1 to enter the lytic or lysogenic pathway.     

Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites.

The NagC repressor controls the expression of the divergently transcribed nagE-nagBACD operons involved in the uptake and degradation of the amino sugars, N-acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN). The glmUS operon, encoding proteins necessary for the synthesis of GlcN (glmS) and the formation of UDP-GlcNAc (glmU), is transcribed from two promoters located upstream of glmU. In the absence of amino sugars both promoters are active. However, in the presence of GlcNAc, the glmU proximal promoter, P1, is inactive while the upstream promoter, P2, is subject to weak induction. Two binding sites for the NagC repressor are located at -200 and -47 bp upstream of P1. Mutations which prevent NagC binding to either of these sites eliminate expression from the P1 promoter. This shows that binding of NagC is necessary for expression of the glmU P1 promoter and implies that NagC is playing the role of activator for this promoter. Moreover, the location of the distal NagC site suggests that this site is behaving like an upstream activating sequence (UAS).     

The bof gene of bacteriophage P1: DNA sequence and evidence for roles in regulation of phage c1 and ref genes.

The C1 repressor of bacteriophage P1 acts via 14 or more distinct operators. This repressor represses its own synthesis as well as the synthesis of other gene products. Previously, mutation of an auxiliary regulatory gene, bof, has been shown to increase expression of some C1-regulated P1 genes (e.g., ref) but to decrease expression of others (e.g., ban). In this study the bof gene was isolated on the basis of its ability to depress stimulation of Escherichia coli chromosomal recombination by the P1 ref gene, if and only if a source of C1 was present. C1 alone, but not Bof alone, was partially effective. The bofDNA sequence encodes an 82-codon reading frame that begins with a TTG codon and includes the sites of the bof-1(Am) mutation and a bof::Tn5 null mutation. Expression of ref::lacZ and cl::lacZ fusion genes was partially repressed in trans by a P1 bof-1 prophage or by plasmid-encoded C1 alone, which was in agreement with effects on Ref-stimulated recombination and with previous indirect evidence for c1 autoregulation. Repression of both fusion genes by plasmid-encoded C1 plus Bof or by a P1 bof+ prophage was more complete. When the C1 source also included a 0.7-kilobase region upstream from C1 which encodes the coi gene, repression of both c1::lacZ and ref::lacZ by C1 alone or by C1 plus Bof was much less effective, as if Coi interfered with C1 repressor function.     

A bacteriophage P1-encoded modulator protein affects the P1 c1 repression system

Bacteriophage P1 encodes a tripartite immunity system composed of the immC, immI, and immT region. Their basic genetic elements are the c1 repressor of lytic functions, the c4 repressor which negatively regulates antirepressor synthesis, and the bof gene, respectively. The function of the latter will be described here. We have cloned and sequenced the bof gene from P1 wild type and a P1 bof amber mutant. Based on the position of a TAG codon of the bof amber mutant the bof wild type gene was localized. It starts with a TTG codon, comprises 82 codons, and is preceded by a promoter structure. The bof protein (Mr = 7500) was overproduced in Escherichia coli from a bof recombinant plasmid and was purified to near homogeneity. The N-terminal amino acids predicted from the DNA sequence of the bof gene were confirmed by sequence analysis of the bof protein. Using a DNA mobility shift assay, we show that bof protein enhances the binding of c1 repressor to the operator of the c1 gene. In accordance with this result, in transformants of Escherichia coli, containing both a bof- and a c1-encoding plasmid, c1 expression is down-regulated. We conclude that bof acts as a modulator protein in the repression of a multitude of c1-controlled operators in the P1 genome.     

Determining the DNA sequence elements required for binding integration host factor to two different target sites.

Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system. IHF was unable to act as a repressor in the challenge-phage assay at H' sites containing the core consensus element but lacking the dA+dT-rich element. This indicates that both elements are required for IHF to bind to the H' site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site, which lacks an upstream dA+dT-rich region. Fifty mutants that decreased or eliminated IHF binding to the H1 site were isolated. Sequence analysis showed changes in the bases in the core consensus element only, further indicating that this determinant is sufficient for IHF binding to the H1 site. We found that placement of a dA+dT-rich element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT-rich element enhances IHF binding.