See also transcription

Comparative genome analyses indicate that increases in gene number do not account for increases in morphological and behavioral complexity. For example, the nematode worm has about 20,000 genes, but lacks the full range of cell types and tissues seen in the fruitfly which contains fewer than 14,000 genes. How has evolutionary diversity arisen? Evidence suggests that organismal complexity is not related to gene number but rather from progressively more elaborate regulation of gene expression.

There are many ways in which a relatively small number of genes can be exploited so as to generate more complex organisms over evolutionary time. Two mechanisms are alternative splicing-the production of different RNA species form a given gene during mRNA splicing- and DNA rearrangement, where genes themselves are rearranged during cellular differentation, as used to generate diversity in mammalian immune systems.  Another way is greater elaboration of regulatory DNA sequences, which control the expression of nearby genes, and increased complexity in the multiprotein transcription compelxes that regulate gene expression.

Transcription is initiated at gene promoters, but many classes of transcriptional regulators, including DNA-binding transcription factors, coactivators, corepressors and proteins that alter epigenetic modifications of DNA and nucleosomal histones, combine to influence the function of minimal promoters. These transcriptional regulators act through a variety of DNA regulatory elements including enhancers, silencers and insulators, which are often located far from the gene promoters. DNA-based regulatory modules may be located in the introns of the regulated gene, a few hundred base pairs to hundreds of kilobases 5′ or 3′ of the gene, or even in the introns of a neighboring irrelevant gene.

Genome sequence comparisons are used to detect noncoding genomic regions that have been evolutionarily conserved, presumbly to maintain come critical biological function. Such regions, called conserved noncoding sequences (CNSs), often correspond to dispersed transcriptional regulatory elements. CNS may also correspond to loci for noncoding RNAs or provide signals necessary for regulated mRNA splicing. Knowledge of the genomic location of CNS regions facilitates analysis of their biological functions. The regions can be cloned and tested in cell based reporter assay or deleted or mutated in their natural genomic context or in the context of bacterial or yeast artificial chromosome transgenes. Different cell types expressing the same gene choose different subsets of the CNS regions as  Dnase I hypersensitive sites  implying that the evolutionary conservation of these regions derives not from their simultaneous participation in gene expression in all expressing cell types but rather from their specialized roles as protein binding regulatory elements in individual cell types.

Typically each DNA regulatory element binds a different subset of transcriptional regulators and thus is independently controlled, imparting modularity to gene regulation.

Control of Transcription:

Gene DNAs and Regulatory Proteins:

Alterations in Chromatin Stucture as a transcriptional control

Transcription Attenuation: In this type of transcriptional control RNA synthesis occurs but for some reason transcription is terminated prematurely. This could happen, for example, where the nascent RNA chain adopts a structure that causes it to interact with RNA polymerase so as to abort transcription. HIV uses a form of transcription attenuation in its life cycle.

Alternative RNA Splicing: is used by many organisms to make different polypeptide chains from the same gene. The regulation of RNA splicing can generate different versions of a protein in different cell types. Recent estimates from expressed sequence tag (EST) studies indicate that 40-60% of human genes are alternatively spliced, and in many cases alternative isoforms result in proteins of distinct function. For example, isoforms generated by alternative splicing may show change or loss of specific function(s) or localization of the respective product, or even a gain of a novel unexpected function (Sykorova, Biol. Cell, 2009, p. 381, 2nd column, lines 1-6).

Biologically relevant isoform differences range form subtle, such as a few nucleotides at an alternative 5′ or 3′ splice site, to skipping several consecutive exons. Variant isoforms can be specific to tissue types or developmental stages and are involved in a large number of normal cellular functions. Defects in splicing also account for a substantial fraction of human genetic disease.

A human example of tissue specific alternative splicing involves proteins produced by the thyroid gland and the hypothalamus. These two organs produce two distinct hormones: calcitonin and CGRP (calcitonin-gene-related peptide) form one gene. Calcitonin controls calcium uptake and the balance of calcium in tissues such as bones and teeth. CGFP is involved in a number of neural and endocrine functions. Despite their different physiological roles, they are produced form the same primary transcript. Whether calcitonin or CGRP is produced depends on different splicing factors in the hytroid and the hypothalamus.

RNA 3′ End Cleavage: In eukaryotes, the 3′ end of a mRNA results from the termination of RNA synthesis by the RNA polymerase and then a cleavage reaction. Cells can regulate where this cleavage event occurs so as to change, for example, the C terminus of the resultant protein. During the development of B lymphocytes, for example, the antibody it produces is anchored in the PM where it serves as a receptor for antigen. Antigen stimulation causes B lymphocytes to multiply and to begin secreting their antibody which is identical to the membrane bound form except for a shorter string of hydrophilic amino acids instead of the long string of hydrophobic amino acids on the membrane bound form. This change is generated through a change in the site of cleavage at the 3′ end. In unstimulated B lymphocytes, the first cleavage poly A addition site encountered by an RNA polymerase is suboptimal and usually skipped leading to production of the longer transcript. When antibody stimulation causes an increase in CSTF concentration, cleavage occurs at the suboptimal site which results in a shorter transcript but one which includes some intron sequence having which encodes the hydrophilic portion (since early cleavage also occurred in front of the 3′ splice site necessary for intron removal).

RNA Editing: alters the nucleotide sequences of mRNA transcripts once they are transcribed. In mammals, for example, there can be enzymatic deamination of adenine to produce inosine which can change the splicing pattern in the RNA or even change the meaning of codons since inosine can base pair with cytosine. Editing is carried out by protein enzymes.

Post Transcriptional Controls

Translational Repressors: Bacteria have a Shine-Dalgarno sequence upstream of the initiating AUG that base pairs with the 16S RNA in the small ribosomal subunit, correctly positioning the initiating AUG codon in the ribosome. Many bacterial mRNAs have specific translational repressor proteins that can bind in this vicinity and inhibit translation of only that species of mRNA. Eucaryotic mRNA do not contain a Shine-Dalgarno sequence but rather use the 5′ cap of the mRNA by the small ribosomal subunit to start scanning for an initiating AUG codon. Some repressor proteins bind to the 5′ cap and inhibit translation initiating.

Phosphorylation of Initiation Factor: The initiation factor eIF-2 for translation in eukaryotes binds very tightly to GDP such that another initiating factor protein is required to cause GDP release so that a new GTP molecule can bind and eIF-2 can be reused. The reuse of eIF-2 is inhibited when it is phosphoyrlated. Regulation of the level of active eIF-2 is important in mammalian cells that allows them to enter a nonproliferating resting state called G0.

mRNA degradation: Most mRNAs in bacteria are very unstable, having a half life of about 3 minutes. Many mRNAs in eucaryotic cells that code for regulatory proteins also have half lives of 30 minutes or less. Exonucleases are responsible for the rapid destruction of bacterial mRNAs. In eucaryotes, the poly A tails are gradually shortened by an exonuclease once the mRNAs enter the cytoplasm. Once a critical threshold is reached, the 5′ cap is removed and the RNA degraded.

Some mRNA also have sequences in their 3′ UTR region that serves as recognition sequences for endonucleases to cleave the poly A tail in one step. For example, many contain AU-rich elements (AREs) in their 3′ untranslated region (UTR). These AREs characteristically comprise clusters of the motif AUUUA and confer instability. Activity in the p38 MAPK pathway can counter this destabilizing effect and so stabilizes the mRNA in question.

mRNAs containing TTATTTAT is detected in the 3’UTR of many cytokine genes: TNF?, TNF?, IL-1alpha, IL-1beta, M-CSF, IFNalpha, IFNbeta, c-fos. This element is important for enhanced turnover of cytokine mRNAs.

See Immunol. 2005 Jan 15;174(2):953-61; Arthritis Res Ther. 2004;6(6):248-64; and Science. 1998 Aug 14;281(5379):1001-5.

RNA interference (RNAi): see outline