Brigitte von Wilcken-Bergmann

Four dimers of λ repressor bound to two suitably spaced pairs of λ operators form octamers and DNA loops over large distances

Abstract Transcription factors that are bound specifically to DNA often interact with each other over thousands of base pairs [1,2]. Large DNA loops resulting from such interactions have been observed in Escherichia coli with the transcription factors deoR [3] and NtrC [4], but such interactions are not, as yet, well understood. We propose that unique protein complexes, that are not present in solution, may form specifically on DNA. Their uniqueness would make it possible for them to interact tightly and specifically with each other. We used the repressor and operators of coliphage λ to construct a model system in which to test our proposition. λ repressor is a dimer at physiological concentrations, but forms tetramers and octamers at a hundredfold higher concentration. We predict that two λ repressor dimers form a tetramer in vitro when bound to two λ operators spaced 24 bp apart and that two such tetramers interact to form an octamer. We examined, in vitro , relaxed circular plasmid DNA in which such operator pairs were separated by 2,850 bp and 2,470 bp. Of these molecules, 29% formed loops as seen by electron microscopy (EM). The loop increased the tightness of binding of λ repressor to λ operator. Consequently, repression of the λ PR promoter in vivo was increased fourfold by the presence of a second pair of λ operators, separated by a distance of 3,600 bp.

The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding

We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix. We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator. We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix. Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator. They further suggest that residue 9 of the recognition helix (asparagine 25) interacts non-specifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3.

CO-OPERATIVE BINDING OF TWO TRP REPRESSOR DIMERS TO ALPHA - OR BETA -CENTRED TRP OPERATORS

The α‐centred trp operator binds one dimer of the Trp repressor, whereas the β‐centred trp operator binds two dimers of the Trp repressor (Carey et al., 1991; Haran et al., 1992). The Trp repressor with a Tyr‐Gly‐7 substitution binds almost as well as the wild‐type Trp repressor to the α‐centred trp operator, but it does not bind to the β‐centred trp operator. This confirms that Tyr‐7 is involved in the interaction between Trp repressor dimers, as seen in the crystal structure (Lawson and Carey, 1993). Further experiments with a‐centred trp operator variants showed that positions 1 of the a‐centred trp operators play a crucial role in tetramerisation. The two innermost base pairs of the α‐centred trp operator are not involved in contacts with the dimer of the Trp repressor binding to it. However, substitutions in these positions (T‐A to G‐T) effectively transform the α‐centred trp operator into a β‐centred trp operator, and thus encourage the binding of two Trp repressor dimers to this operator. Finally, we demonstrate, with suitable heterodimers, that one subunit of each dimer suffices to bind to a β‐centred trp operator.

Mutants with substitutions for Glu171 in the catabolite activator protein (CAP) of Escherichia coli activate transcription from the lac promoter

Single amino acid substitutions for residue Glu171 in helix E of the catabolite gene activator protein (CAP) of Escherichia coli have been reported to abolish activation of transcription without impairing binding to the CAP site of the lac promoter. The negative charge of Glu171 was proposed to transmit the activating signal from CAP to RNA polymerase. However, this idea has been challenged by later work. We set up a system to re-examine this issue. We analysed the ability of mutant CAP-E 171 L and CAP-E 171 K proteins to bind a nearconsensus CAP site in vivo and found it to be diminished fourfold relative to wild type in each case. Activation of lac transcription by these mutant proteins remains the same as with wild-type CAP. Thus our results confirm that Glu171 in helix E of CAP is not involved directly in the activation of transcription. Yet CAP-E171K does not activate transcription as well as wild-type CAP under all circumstances. Possible reasons for this absence of activation are discussed.

Expression of adenovirus type 12 Elb 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::β-galactosidase fusion protein

DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli.

Searching for the Code of Ideal Protein-DNA-Recognition

A system is described which allows testing of specific protein-DNA-interactions. The system consists of two mutually compatible plasmids carrying different origins of replication and resistance markers. One plasmid carries a lac I gene in which the DNA-recognizing domain has been replaced by synthetic DNA saturated with restriction sites. The other carries a lac P Z unit in which the natural operator has been deleted and replaced by a unique restriction site. Into this restriction site any operator can be cloned.