Question

You are studying a new protein called protein Z that you think is a gene-specific transcription factor that enhances expression of genes that are involved in metabolism. Using an EMSA, you have already shown that protein Z binds to enhancers of a group of genes that code for proteins involved in metabolism. In order for a gene-specific transcription factor to be involved in regulating transcription of a gene that may be located far away, what protein does the gene-specific transcription factor need to interact with? What molecular technique would you use to test whether or not protein Z interacts with this protein?

Answer 1

Nutritional factors can influence virtually every aspect of the functioning of the human organism. This influence extends to the realm of gene expression. By influencing gene expression in specific tissues of the organism, nutritional factors help to adapt the organism to changes in the environment. This review will describe the technologies that have emerged for analyzing the effects of nutrition on gene expression, with particular emphasis on the process of gene transcription and its control. Several model systems then will be described in which changes in specific gene expression in response to nutritional factors have been elucidated and efforts to understand the molecular basis of these changes have been made. While this field is still in its infancy, the pace of change suggests that great strides will soon be forthcoming in understanding these important mechanisms and how they may relate to human health and disease.

Gene expression refers to those processes by which the genetic information stored in the DNA is converted into proteins (including enzymes) within the cell. This is a multistep process that involves gene transcription, mRNA processing (capping, splicing, and polyadenylation), and mRNA transport and translation. Each of these processes in turn involves a complex series of biochemical events. Consequently, control of gene expression can be exerted at many different sites in the cell, and in fact, examples of regulation occurring at each step of this pathway have been elucidated. Such complexity of control is undoubtedly critical to the fine tuning of cellular function within the context of the overall organism. Despite this richness in terms of potential sites of control, it is clear that transcriptional regulation and, more specifically, control of transcriptional initiation provide the most commonly employed site for regulation. Given the importance of transcriptional regulation, much attention has been focused on this process in the past decade and much has been learned. Hence, this chapter will focus primarily on regulation occurring at the level of gene transcription.

Transcription of protein-coding genes in all eucaryotes is performed by the enzyme RNA polymerase II. However, this enzyme lacks the inherent ability to recognize the proper site in DNA for initiation of transcription. Rather, RNA polymerase II functions together with a battery of general ''transcription factors" to perform these processes (for review, see Zawel and Reinberg, 1993). To date, six factors essential to the process of promoter selection and initiation have been identified. These factors, together with RNA polymerase II, recognize specific sequences in the DNA helix at the initiation site that are frequently termed the basal promoter site. The most commonly recognized of these signals is the TATA box, a 7 base pair (bp)-conserved sequence occurring approximately 30 bp upstream from (to the 5' end of) the site of initiation in many genes. Other less-conserved signals at the site of initiation also play a role in the site selection. These sequences are recognized by general transcription factors to initiate assembly of the RNA transcription complex.

Although RNA polymerase II in combination with the general transcription factors is competent to recognize and initiate transcription from the basal promoter, the rate of this process is very low. To achieve effective production of mRNA, other transcription factors need to be brought into play. Because these transcription factors only function on a limited set of genes, they are termed specific transcription factors. There are in excess of 100 such factors present in any particular cell. These specific transcription factors function by binding to specific DNA sequences in the vicinity of the basal promoter site. Many times these sites are located immediately upstream of the basal promoter, within 100 to 200 bp of the initiation site. Other times these factors can function by binding at sites that can be several thousand base pairs removed from the initiation site in regions known as enhancers. These sites serve to localize the specific transcription factors in proximity to the basal transcriptional machinery. The factors in turn influence the rate of initiation, presumably by making protein-protein contacts with RNA polymerase II or its associated factors (Choy and Green, 1993). These contacts are thought to alter the stability or kinetics of initiation-complex formation to stimulate transcription. Thus, the rate of initiation from any specific promoter in any particular cell is determined in large part by the qualitative and quantitative nature of the binding sites for specific transcription factors present on the gene and the concentration and activity of the corresponding transcription factors present in the cell. Regulation can occur by controlling the activity of these specific transcription factors. Thus, understanding the control of transcriptional initiation requires the elucidation of the regulatory sequences present for binding specific transcription factors and the elucidation of the nature of the factors that bind to these sites. Technologies for unraveling these components have developed in the past decade and are essential tools in the arsenal of the molecular biologist.