Question

- Know the different processes of Eukaryotic Regulation (i.e. Chromatin Structure, Transcriptional regulation.

Answer 1

Regulation of Transcription in Eukaryotes

Although the control of gene expression is far more complex in eukaryotes than in bacteria, the same basic principles apply. The expression of eukaryotic genes is controlled primarily at the level of initiation of transcription, although in some cases transcription may be attenuated and regulated at subsequent steps. As in bacteria, transcription in eukaryotic cells is controlled by proteins that bind to specific regulatory sequences and modulate the activity of RNA polymerase. The intricate task of regulating gene expression in the many differentiated cell types of multicellular organisms is accomplished primarily by the combined actions of multiple different transcriptional regulatory proteins. In addition, the packaging of DNA into chromatin and its modification by methylation impart further levels of complexity to the control of eukaryotic gene expression.

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cis-Acting Regulatory Sequences: Promoters and Enhancers

As already discussed, transcription in bacteria is regulated by the binding of proteins to cis-acting sequences (e.g., the lac operator) that control the transcription of adjacent genes. Similar cis-acting sequences regulate the expression of eukaryotic genes. These sequences have been identified in mammalian cells largely by the use of gene transfer assays to study the activity of suspected regulatory regions of cloned genes (Figure 6.18). The eukaryotic regulatory sequences are usually ligated to a reporter gene that encodes an easily detectable enzyme. The expression of the reporter gene following its transfer into cultured cells then provides a sensitive assay for the ability of the cloned regulatory sequences to direct transcription. Biologically active regulatory regions can thus be identified, and in vitromutagenesis can be used to determine the roles of specific sequences within the region.

Figure 6.18

Identification of eukaryotic regulatory sequences. The regulatory sequence of a cloned eukaryotic gene is ligated to a reporter gene that encodes an easily detectable enzyme. The resulting plasmid is then introduced into cultured recipient cells by transfection. (more...)

Genes transcribed by RNA polymerase II have two core promoter elements, the TATA box and the Inr sequence, that serve as specific binding sites for general transcription factors. Other cis-acting sequences serve as binding sites for a wide variety of regulatory factors that control the expression of individual genes. These cis-acting regulatory sequences are frequently, though not always, located upstream of the TATA box. For example, two regulatory sequences that are found in many eukaryotic genes were identified by studies of the promoter of the herpes simplex virus gene that encodes thymidine kinase (Figure 6.19). Both of these sequences are located within 100 base pairs upstream of the TATA box: Their consensus sequences are CCAAT and GGGCGG (called a GC box). Specific proteins that bind to these sequences and stimulate transcription have since been identified.

Figure 6.19

A eukaryotic promoter. The promoter of the thymidine kinase gene of herpes simplex virus contains three sequence elements upstream of the TATA box that are required for efficient transcription: a CCAAT box and two GC boxes (consensus sequence GGGCGG). (more...)

In contrast to the relatively simple organization of CCAAT and GC boxes in the herpes thymidine kinase promoter, many genes in mammalian cells are controlled by regulatory sequences located farther away (sometimes more than 10 kilobases) from the transcription start site. These sequences, called enhancers, were first identified by Walter Schaffner in 1981 during studies of the promoter of another virus, SV40 (Figure 6.20). In addition to a TATA box and a set of six GC boxes, two 72-base-pair repeats located farther upstream are required for efficient transcription from this promoter. These sequences were found to stimulate transcription from other promoters as well as from that of SV40, and, surprisingly, their activity depended on neither their distance nor their orientation with respect to the transcription initiation site (Figure 6.21). They could stimulate transcription when placed either upstream or downstream of the promoter, in either a forward or backward orientation.

Figure 6.20

The SV40 enhancer. The SV40 promoter for early gene expression contains a TATA box and six GC boxes arranged in three sets of repeated sequences. In addition, efficient transcription requires an upstream enhancer consisting of two 72-base-pair (bp) repeats. (more...)

Figure 6.21

Action of enhancers. Without an enhancer, the gene is transcribed at a low basal level (A). Addition of an enhancer, E—for example, the SV40 72-base-pair repeats—stimulates transcription. The enhancer is active not only when placed just (more...)

The ability of enhancers to function even when separated by long distances from transcription initiation sites at first suggested that they work by mechanisms different from those of promoters. However, this has turned out not to be the case: Enhancers, like promoters, function by binding transcription factors that then regulate RNA polymerase. This is possible because of DNA looping, which allows a transcription factor bound to a distant enhancer to interact with RNA polymerase or general transcription factors at the promoter (Figure 6.22). Transcription factors bound to distant enhancers can thus work by the same mechanisms as those bound adjacent to promoters, so there is no fundamental difference between the actions of enhancers and those of cis-acting regulatory sequences adjacent to transcription start sites. Interestingly, although enhancers were first identified in mammalian cells, they have subsequently been found in bacteria—an unusual instance in which studies of eukaryotes served as a model for the simpler prokaryotic systems.

Figure 6.22

DNA looping. Transcription factors bound at distant enhancers are able to interact with general transcription factors at the promoter because the intervening DNA can form loops. There is therefore no fundamental difference between the action of transcription (more...)

The binding of specific transcriptional regulatory proteins to enhancers is responsible for the control of geneexpression during development and differentiation, as well as during the response of cells to hormones and growth factors. One of the most thoroughly studied mammalian enhancers controls the transcription of immunoglobulingenes in B lymphocytes. Gene transfer experiments have established that the immunoglobulin enhancer is active in lymphocytes, but not in other types of cells. Thus, this regulatory sequence is at least partly responsible for tissue-specific expression of the immunoglobulin genes in the appropriate differentiated cell type.

An important aspect of enhancers is that they usually contain multiple functional sequence elements that bind different transcriptional regulatory proteins. These proteins work together to regulate gene expression. The immunoglobulin heavy-chain enhancer, for example, spans approximately 200 base pairs and contains at least nine distinct sequence elements that serve as protein-binding sites (Figure 6.23). Mutation of any one of these sequences reduces but does not abolish enhancer activity, indicating that the functions of individual proteins that bind to the enhancer are at least partly redundant. Many of the individual sequence elements of the immunoglobulin enhancer by themselves stimulate transcription in nonlymphoid cells. The restricted activity of the intact enhancer in B lymphocytes therefore does not result from the tissue-specific function of each of its components. Instead, tissue-specific expression results from the combination of the individual sequence elements that make up the complete enhancer. These elements include some cis-acting regulatory sequences that bind transcriptional activators that are expressed specifically in B lymphocytes, as well as other regulatory sequences that bind repressors in nonlymphoid cells. Thus, the immunoglobulin enhancer contains negative regulatory elements that inhibit transcription in inappropriate cell types, as well as positive regulatory elements that activate transcription in B lymphocytes. The overall activity of the enhancer is greater than the sum of its parts, reflecting the combined action of the proteins associated with each of its individual sequence elements.

chromatin structure

Chromatin is comprised of histones and DNA: 147 base pairs of DNA wraps around the 8 core histones to form the basic chromatin unit, the nucleosome.

The function of chromatin is to efficiently package DNA into a small volume to fit into the nucleus of a cell and protect the DNA structure and sequence. Packaging DNA into chromatin allows for mitosis and meiosis, prevents chromosome breakage and controls gene expression and DNA replication.