A whole genome approach to in vivo DNA-protein interactions in E. coli.

The increasingly rapid pace at which genomic DNA sequences are being determined has created a need for more efficient techniques to determine which parts of these sequences are bound in vivo by the proteins controlling processes such as gene expression, DNA replication and chromosomal mechanics. Here we describe a whole-genome approach to identify and characterize such DNA sequences. The method uses endogenous or artificially introduced methylases to methylate all genomic targets except those protected in vivo by protein or non-protein factors interfering with methylase action. These protected targets remain unmethylated in purified genomic DNA and are identified using methylation-sensitive restriction endonucleases. When the method was applied to the Escherichia coli genome, 0.1% of the endogenous adenine methyl-transferase (Dam methylase) targets were found to be unmethylated. Five foreign methylases were examined by transfection. Database-matched DNA sequences flanking the in vivo-protected Dam sites all fell in the non-coding regions of seven E. coli operons (mtl, cdd, flh, gut, car, psp and fep). In the first four operons these DNA sequences closely matched the consensus sequence that binds to the cyclic AMP-receptor protein. The in vivo protection at the Dam site upstream of the car operon was correlated with a downregulation of car expression, as expected of a feedback repressor-binding model.     

Neutron scattering studies of lac repressor

The lac repressor, a tetrameric protein of identical subunits [molecular weight (MW) 4 x 38,500], interacts specifically with the lac operator, preventing the expression of the structural genes of the lac operon. The presence of an inducer (such as isopropyl-beta-D-thiogalactoside; IPTG) which binds to the repressor, prevents operator binding by lowering the association constant by a factor of 10(3) (ref. 3). Genetic and biochemical analysis have shown that the major part--if not all--of the binding site for the lac operator is located in the 60 N-terminal residues of the protein. In certain conditions, limited trypsinolysis of the protein yields four N-terminal 'headpieces' each containing 51 or 59 residues, and a tetrameric core with full inducer binding activity. It was shown recently that this headpiece is able to bind nucleic acids, and interacts with the lac operator, giving the same pattern of sensitivity with respect to the methylation of the bases as does the intact repressor. We are studying the interaction of lac repressor with DNA by neutron scattering using contrast variation and discuss here measurements on the protein, its tryptic core and their complexes with IPTG. Our results demonstrate that the headpieces are located far (67 +/- 10 A) from the centre of mass of a somewhat elongated core, and that the inducer does not significantly change the radius of gyration of the protein.     

Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures.

We have isolated a set of ligand-binding DNA sequences from a large pool of random sequence DNAs by selection and amplification in vitro, using similar methods to those described for the isolation of ligand-binding RNAs. The ligand-DNA interactions are both sequence- and ligand-specific, and are dependent on proper folding of the single-stranded DNA. Some ligands led to the isolation of more DNA sequences than RNA sequences, and vice versa. Analysis of individual sequences reveals that ligand binding is DNA-specific; RNAs of identical sequence could not interact with the same ligands. Ligand-binding DNAs might be more suitable than RNAs as potential pharmacological reagents because of the greater stability of DNA. The apparent primacy of RNA in the early evolution of life may have been due to its availability rather than to its functional superiority.     

Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA-binding state

A central question arising from the model of eukaryotic gene regulation by steroid hormone receptors is whether or not proteins represent pre-existing gene regulatory proteins that are activated on exposure to the extracellular signal. It has been generally believed that the ligand-binding of steroid hormone receptors triggers an allosteric change in receptor structure, manifested by an increased affinity of the receptor for DNA in vitro and nuclear target elements in vivo, as monitored by nuclear translocation. But this model has been challenged by recent reports indicating that glucocorticoid and progesterone receptors bind specifically in vitro to target DNA sequences even in the absence of hormone. On the other hand, it appears that the hormone induces protection in vivo of the glucocorticoid response element of the tyrosine amino transferase gene. Here we show that under conditions permitting minimal in vitro manipulation, the steroid-free glucocorticoid receptor in crude cytosol associates with the hsp90 heat shock protein (relative molecular mass Mr approximately equal to 90,000) to form a large 300K complex, rather than the 94K liganded receptor monomer. More importantly, we have developed an assay to demonstrate the requirement of hormone to dissociate the 300K complex by heat treatment. Specific DNA-binding activity of the receptor becomes apparent in this process, showing that DNA binding occurs but is inhibited in the large heteromeric complex. We propose a model in which receptor function is repressed by association of the receptor with hsp90. Dissociation of this complex is induced by the binding of steroid and is apparently an irreversible process.     

Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase

In physiological settings, nucleic-acid translocases must act on substrates occupied by other proteins, and an increasingly appreciated role of translocases is to catalyse protein displacement from RNA and DNA. However, little is known regarding the inevitable collisions that must occur, and the fate of protein obstacles and the mechanisms by which they are evicted from DNA remain unexplored. Here we sought to establish the mechanistic basis for protein displacement from DNA using RecBCD as a model system. Using nanofabricated curtains of DNA and multicolour single-molecule microscopy, we visualized collisions between a model translocase and different DNA-bound proteins in real time. We show that the DNA translocase RecBCD can disrupt core RNA polymerase, holoenzymes, stalled elongation complexes and transcribing RNA polymerases in either head-to-head or head-to-tail orientations, as well as EcoRI(E111Q), lac repressor and even nucleosomes. RecBCD did not pause during collisions and often pushed proteins thousands of base pairs before evicting them from DNA. We conclude that RecBCD overwhelms obstacles through direct transduction of chemomechanical force with no need for specific protein-protein interactions, and that proteins can be removed from DNA through active disruption mechanisms that act on a transition state intermediate as they are pushed from one nonspecific site to the next.     

Expression of the E. coli uvrA gene is inducible

UvrA+-dependent excision repair is one of the most important systems in Escherichia coli for repairing UV-induced pyrimidine dimers and a variety of other forms of DNA damage. The uvrA protein acts in conjunction with the uvrB and uvrC gene products to introduce a nick at the of a DNA lesion and thus initiate the repair process. We have recently used the Mud(Ap, lac) operon fusion vector to identify a set of genes whose expression is induced by DNA damage. One Mud(Ap, lac) insertion mapped at the uvrA locus and made the cells sensitive to UV light. In this fusion strain, beta-galactosidase expression was induced by DNA-damaging agents in a recA+lexA+-dependent fashion. We were surprised by this result because uvrA+-dependent excision repair is observed both in cells in which protein synthesis has been inhibited and in recA- and lexA- cells, findings which have led to the conclusion that the uvrA gene product is constitutively expressed and not under the control of the complex recA+lexA+ regulatory circuitry (see below). We have investigated this possibility further and describe here the generation and characterization of a set of fusions of the lac genes to the promoter of the uvrA gene. We confirm that the uvrA gene product is induced by DNA damage in a recA+lexA+-dependent fashion.     

TIAR通过在DNA和RNA之间的穿梭机制调控DNA的转录活性

应用体外转录模型,即通过构建T7 及T7 TIAR 的碱基序列及部分退火的双链模型,合成了一段ODN,其包含单链的TIAR结合位点和1个T7的起始位点.通过进行RNA转录分析,TIAR的结合和替代实验,证明了TIAR可与富含T的单链DNA结合,并且TIAR与DNA的结合可因DNA的转录活性而解离.这一发现为TIAR可在DNA与RNA之间穿梭提供了证据.     

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes

Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.     

Optimality and evolutionary tuning of the expression level of a protein

Different proteins have different expression levels. It is unclear to what extent these expression levels are optimized to their environment. Evolutionary theories suggest that protein expression levels maximize fitness, but the fitness as a function of protein level has seldom been directly measured. To address this, we studied the lac system of Escherichia coli, which allows the cell to use the sugar lactose for growth. We experimentally measured the growth burden due to production and maintenance of the Lac proteins (cost), as well as the growth advantage (benefit) conferred by the Lac proteins when lactose is present. The fitness function, given by the difference between the benefit and the cost, predicts that for each lactose environment there exists an optimal Lac expression level that maximizes growth rate. We then performed serial dilution evolution experiments at different lactose concentrations. In a few hundred generations, cells evolved to reach the predicted optimal expression levels. Thus, protein expression from the lac operon seems to be a solution of a cost-benefit optimization problem, and can be rapidly tuned by evolution to function optimally in new environments.