The British Biophysical Society

Regulation of Gene Expression

Andrew Lane

 

Gene expression is the transfer of the information encoded within the genome into proteins via an intermediate message, mRNA. Each stage involves an amplification step; multiple copies of mRNA are transcribed from the DNA, and a large number of protein molecules are translated from each mRNA. Each step is tightly controlled by the action of proteins by forming specific protein-DNA complexes, ensuring that the correct amount of protein is made for a given cell type, according to its current metabolic needs.

It may also be desirable to control gene expression externally, for instance to correct some malfunction in the cell, or as a means of killing foreign cells (bacteria, viruses etc). For example, inhibition of specific proteins (usually enzymes) by drugs is a long-standing goal of the pharmaceutical industry. The optimization of a drug's efficacy requires detailed knowledge of the properties of the protein, its structure, and how it interacts with the drug. This is in the realm of structural biology, and is a fundamental component in RATIONAL DRUG DESIGN.

In addition to inhibiting particular gene products, it may be more efficient to interfere with the earlier stages of its production, either by inhibiting the translation of the mRNA, which blocks one amplification step, or at the level of the gene itself, which then blocks both amplification steps. This would be desirable as it can limit the dose of the drug required to attain the desired response, and thereby perhaps reduce side effects.

It is possible to design drugs that bind to DNA, based on the structural properties of DNA sequences as determined by X-ray Crystallography and NMR SPECTROSCOPY. The complex formed between the drug and DNA is then unable to be transcribed. Most of the small molecule drugs bind to the minor groove of the DNA, and make intimate contacts with the walls of the groove, and numerous hydrogen bonding and electrostatic interactions with the bases and the phosphate backbone. An example is the structure of the propamidine-d(CGCAAATTTGCG)2 complex (below).

The solution structure of the complex was determined by NMR and molecular modelling methods (stick model (left) and Space-filling model (right)). This drug molecule (in yellow) specifically binds to AATTT sequences in the minor groove, and fits snugly without distorting the DNA. Drugs of this class are used to treat opportunisitic infections in immunocompromised patients.

It is also possible to get very high sequence specificity by using oligonucleotides that recognize DNA duplexes by hydrogen bonding to the DNA in its major groove, forming a DNA triple helix. This is the so-called antigene approach. The improvement of the actual sequence selection and overall affinity requires detailed structural knowledge of the DNA target and the complex, as well as information about how strongly the components interact (thermodynamics), and how fast they form the inhibiting complex (kinetics).

The structure was determined in solution by NMR methods (Space-filling model (left) and stick model (right)). The view shows the third (therapeutic) strand filling the major groove of the Watson-Crick duplex, parallel to the purine strand, and making Hoogsteen hydrogen bonds to the purine bases.

In an analogous approach, antisense technology, a DNA oligonucleotide is designed to be complementary to a stretch of a particular mRNA, forming a hybrid RNA.DNA duplex that cannot be translated. The stability and properties of such duplexes must be understood so that optimal antisense DNA can be designed. Further, this hybrid is a substrate for the ubiquitous RNAseH, which cleaves only the RNA strand in DNA.RNA hybrids, thereby regenetrating the antisense oligonucleotide. Therefore the sensitivity of the hybrids to the enzyme also have to be taken into account, requiring in addition to structural information, thermodynamics and kinetics, the detailed enzymology of RNaseH activityon the different hybrid sequences.

Andrew Lane is Professor of Medicine and Joint Professor of Chemistry at the University of Louisville, where he also holds the James Graham Brown Endowed Chair of Structural Biology. He is also directory of the James Graham Brown Cancer Center NMR Facility and is on the Editorial Boards of Nucleic Acids Research and Journal of Structural and Functional Genomics.