Regulation of Transcription in Eukaryotes

While both prokaryotic and eukaryotic genes are regulated by activators and repressors, enhancer elements are unique to eukaryotic genes and can profoundly increase the rate of transcription. These elements are located at a variable distance from the basic promoter itself, can be present both upstream or downstream to the promoter, and, in fact, can even be within the transcription unit. One unexpected feature is that they can function in either orientation and can activate any promoter located in the vicinity.

Upstream activating sequences (UAS) have been identified in yeast and are analogous to enhancers in the mammalian genes. Based on the known properties of enhancers, it appears that the presence of these sequences affects chromatin structure and/or the helical structure of the DNA template itself. Further studies are needed to test other possibilities as well, e.g., whether the enhancer provides an entry point for the transcription complex or is needed to place the template at the nuclear matrix where transcription takes place.

Positive and negative regulation of prokaryotic genes is achieved by binding of activators and repressors, respectively, to their cognate binding sites in the genes. Downregulation is more common, at least in E. coli, than positive regulation. In fact, the same protein can provide dual functions in a few cases, depending on the location of the sequence motif.

In contrast, because of the complexity of chromatin structure and genomic organization development, differentiation, and cell cycle-specific synthesis of proteins, regulation of eukaryotic genes is extremely complex. This is evident from the large number of families of regulatory trans-acting factors which recognize similar if not identical sequence motifs in different genes. Sometimes, these factors have a distinct modular structure—one module for binding to target DNA sequence and another for interaction with components of the transcription apparatus.

On top of these complexities, the signal for initiation of transcription may be extracellular, e.g., a growth factor which induces cell proliferation. A highly complex signaling cascade is initiated in response to the first signal. The external ligand first binds to its receptor on the cell surface, followed by internalization of the receptor ligand complex. A series of reversible chemical modification (mostly phosphorylation of the regulatory proteins) finally activates the ultimate transcription factors, which then trigger transcription of target genes.

The unique difference between the eukaryotes and prokaryotes is in the utilization of transcription factors. In bacteria, one factor is usually specific for one gene or one regulatory unit. In eukaryotes, on the other hand, a single factor activates multiple target genes.

Prokaryotic regulatory processes have been elucidated in remarkable detail by utilizing the power of molecular genetics, including “reverse genetics” by which the chromosomal genes in the organism could be mutated at specific sites and the mutant gene products purified and characterized. Furthermore, these genes can be expressed in the episomal state by introducing them into autonomously replicating recombinant plasmids.

Commensurate with the significantly higher complexity and size of the genome and differentiation and developmental stages in metazoans, gene regulation in these organisms is very complex and occurs at many levels. Sets of genes are activated at distinct stages of differentiation and development of multicellular organisms in order to encode proteins which are required for specialized functions of the cells in these stages. In contrast, certain “housekeeping” proteins, including enzymes for metabolism and synthesis of all cellular components (i.e., RNA, DNA, structural proteins, and lipids), as well as enzymes for biosynthetic and degradative pathways, are needed in all cell types and developmental stages. Most somatic cells in adult mammals are nondividing and therefore do not require DNA synthesis machinery. However, all cells require transcription for generating proteins for other cellular functions. Unraveling the molecular mechanisms of regulation is the major focus of current research in molecular biology. The regulatory process is affected by multiple parameters.

Many genes are activated due to external stimuli, e.g., exposure to hormones and growth factors. In these cases the extracellular signal often acts as a ligand to bind to cell surface receptors which activate the trans-acting factor(s) via multiple steps of signal transduction.

1. Regulation of Transcription via Chromatin Structure Modulation in Eukaryotes
The eukaryotic genome is organized at multiple levels, starting with the nucleosome core as described earlier. The nucleosomes are organized in a higher order chromatin structure due to increasing compaction of DNA: from 2-nm-wide naked DNA fiber to metaphase chromosomes of microscopic width. The DNA template has to be accessible to transcription machinery containing RNA polymerase; transcriptionally inactive, highly compacted chromatin maintains its structure by multiple protein–protein and protein–DNA interactions, which are yet to be elucidated. However, it is now clear that at the nucleosome level, it is the strength of interaction between histones and DNA which regulates accessibility of the DNA to the transcription machinery, a process controlled by acetylation and phosphorylation of core histones. Multiple histone acetylases and deacetylases, which are themselves regulated, modulate chromatin structure. As stated previously, large protein complexes named SWI and SNF modulate chromatin structure in an energy-dependent process which may be responsible for the differentiation/ development-dependent turning on or off of specific sets of genes.

2. CpG Methylation-Dependent Negative Regulation of Genes
In addition to histone modification, DNA itself was found to be modified, most commonly by methylation at the C-5 position of cytosine, but only when it is present as a CpG dinucleotide. Such methylation, catalyzed by specific methyltransferases, invariably inhibits gene expression, which was unequivocally established in the genomes during embryonic development. Sets of genes are selectively methylated or demethylated in the CpG sequences, most commonly in the genes’ promoter regions, leading to their activation or repression. Proteins that bind to methylated CpG sequences have been implicated in the control of histone deacetylation, thereby leading to closing of the promoter.