Biomedical subjects

T A Lavoie

Publications and source records attributed to T A Lavoie.

4 recordsLinked to original sources

PubMed1999

Cooperative folding units of escherichia coli tryptophan repressor.

A previously published computational procedure was used to identify cooperative folding units within tryptophan repressor. The theoretical results predict the existence of distinct stable substructures in the protein chain for the monomer and the dimer. The predictions were compared with experimental data on structure and folding of the repressor and its proteolytic fragments and show excellent agreement for the dimeric form of the protein. The results suggest that the monomer, the structure of which is currently unknown, is likely to have a structure different from the one it has within the context of the highly intertwined dimer. Application of this method to the repressor monomer represents an extension of the computations into the realm of evaluating hypothetical structures such as those produced by threading.

Apoproteins↗

PubMed1996

In vivo and in vitro studies of TrpR-DNA interactions.

The binding of tryptophan repressor (TrpR) to its operators was examined quantitatively using in vitro and in vivo methods. DNA sequence requirements for 1:1 and tandem 2:1 (TrpR:DNA) binding in various sequence contexts were studied. The results indicate that the optimal half-site sequence for recognition by one helix-turn-helix motif of one TrpR dimer is 3'CNTGA5'5'GNACT3', consistent with contacts observed by X-ray diffraction analysis of cocrystalline 1:1 and 2:1 complexes. Half-sites can be paired to form a palindrome either by direct abutment, forming the nucleation site for a tandem 2:1 complex, or with an 8-base-pair spacer, forming a 1:1 target. Dimethylsulfate (DMS) methylation-protection footprinting in vitro of 1:1 and 2:1 complexes formed sequentially on the two unequal half-site pairs of the trpEDCBA operator from Serratia marcescens indicated an obligate hierarchy of site occupancy, with one half-site pair serving as the nucleation site for tandem binding. DMS footprinting of Escherichia coli operators in vivo showed that, over a wide range of intracellular TrpR concentration, the trpEDCBA operator is occupied by three repressor dimers, aroH is occupied by two dimers, and the 1:1 binding mode is used on the trpR operator. The coexistence of these distinct occupancy states implies that changes in protein concentration affect only the fractional occupancy of each operator rather than the binding mode, which is determined by the number of half-site sequences present in the operator region. Cooperativity of tandem complex formation measured by gel retardation using a symmetrized synthetic operator containing identical, optimal sites spaced as in natural operators was found to be modest, implying a maximum coupling free energy of approximately -2 kcal/mol. On other sequences the apparent degree of cooperativity, as well as the apparent affinity, varies with sequence and sequence context in a manner consistent with the structural models and which suggests compensation between affinity and cooperativity as a mechanism that allows tolerance of operator sequence variation.

Bacterial Proteins↗

PubMed1995

The DNA-binding domain of the hexameric arginine repressor.

The arginine repressor of Escherichia coli is a classical feedback regulator, signalling the availability of L-arginine inside the cell. It differs from most other bacterial repressors in functioning as a hexamer, but structural details have been lacking and its shares no clear sequence homologies with other transcriptional regulators. Analysis of the amino acid residue sequence and proteolytic cleavage pattern of the repressor was used to identify a region predicted to house the DNA-binding function. When this protein fragment is overexpressed from a clone of the corresponding gene fragment, it represses ornithine transcarbamylase levels in vivo, and binds to the operator DNA in vitro, both in an arginine-independent manner. Sedimentation equilibrium and gel filtration indicate that the purified protein fragment is a monomer in solution. The results thus define the domain organization of the repressor at low resolution, suggesting that the N and C-terminal portions of the polypeptide chain are separated by a structural and functional border that decouples hexamerization and arginine binding from DNA binding.

Allosteric Regulation↗

PubMed1991

How does trp repressor bind to its operator?

Three explanations have been advanced to account for the unexpected absence of direct hydrogen bonds and presence of a buried water layer in the co-crystal-line complex of Escherichia coli trp repressor with DNA. We present results of physical and biochemical measurements that address the testable predictions of each model. We find that the DNA oligomer used for co-crystallization binds to the repressor with high affinity and specificity, and 1:1 stoichiometry, consistent with other evidence that this sequence represents the true operator target for a single repressor dimer. A proposed alternative DNA sequence binds weaker and with higher stoichiometry, consistent with a cooperative binding mode. The operator DNA in solution has a B-form helical structure in the presence and absence of repressor. Affinity of repressor for operator is altered under the conditions used for cocrystal growth.

Bacterial Proteins↗