Gene Regulation

Part III

Mechanism of regulation of gene expression- An overview

• Gene activity is controlled first and foremost at the level of transcription.

• Much of this control is achieved through the interplay between proteins that bind to specific DNA sequences and their DNA binding sites.

• This can have a positive or negative effect on transcription.

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• Transcription control can result in tissue-specific gene expression.

• In addition to transcription level controls, gene expression can also be modulated by :

1. Gene amplification

2. Gene rearrangement

3. Posttranscriptional modifications, and

4. RNA stabilization.

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• Eukaryotes – There are two types of promoters which are:

• Basal promoter or core promoter -These promoters reside within 40 bp upstream of the start site. These promoters are seen in all protein coding genes.

• Upstream promoters - These promoters may lie up to 200bp upstream of the transcriptional initiation site. The structure of this promoter and the associated binding factors keeps varying from gene to gene.

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Transcriptional control: … controlling when and how often a given gene is Transcribed

Genes can be expressed with different efficiencies. Gene A is transcribed and translated much more efficiently than gene B. This allows the amount of protein A in the cell to be much greater than that of protein B.

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Gene Amplification

• Such requirements are fulfilled by amplification of these specific genes.

• Subsequently, these amplified genes, presumably generated by a process of repeated initiations during DNA synthesis, provide multiple sites for gene transcription.

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Gene Amplification

• The gene product can be increased by increasing the number of genes available for transcription of specific molecules

• Among the repetitive DNA sequences are hundreds of copies of ribosomal RNA genes and tRNA genes.

• During early development of metazoans, there is an abrupt increase in the need for ribosomal RNA and messenger RNA molecules for proteins that make up such organs as the eggshell.

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Gene Amplification

• Gene amplification has been demonstrated in patients receiving methotrexate for cancer.

• The malignant cells can develop drug resistance by increasing the number of genes for dihydrofolate reductase, the target of Methotrexate.

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Differences between gene expression in prokaryotes and eukaryotes

Gene regulation is significantly more complex in eukaryotes than in prokaryotes for a number of reasons:

1) First, the genome being regulated is significantly larger

o The E. coli genome consists of a single, circular chromosome containing 4.6 Mb.

o This genome encodes approximately 2000 proteins.

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1) Larger genome

• In comparison, the genome within a human cell contains 23 pairs of chromosomes ranging in size from 50 to 250 Mb.

• Approximately 40,000 genes are present within the human DNA.

• It would be very difficult for a DNA-binding protein to recognize a unique site in this vast array of DNA sequences.

• More-elaborate mechanisms are required to achieve specificity

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2) Different cell types

• Different cell types are present in most eukaryotes.

• Liver and pancreatic cells, for example, differ dramatically in the genes that are highly expressed.

• Different mechanisms are involved in the regulation of such genes.

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3) Absence of operons

• The eukaryotic genes are not generally organized into operons as are there in prokaryotes

• Instead, genes that encode proteins for steps within a given pathway are often spread widely across the genome.

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4) Chromatin structure

• The DNA in eukaryotic cells is extensively folded and packed into the protein-DNA complex called chromatin.

• Histones are an important part of this complex since they both form the structures known as nucleosomes and also contribute significantly into gene regulatory mechanisms.

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5) Uncoupled transcription and translation processes

• In prokaryotes, transcription and translation are coupled processes, the primary transcript is immediately translated.

• The transcription and translation are uncoupled in eukaryotes, eliminating some potential gene-regulatory mechanisms.

• The primary transcript in eukaryotes undergoes modifications to become a mature functional m RNA.

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Enhancers and Repressors

• Enhancer elements are DNA sequences, although they have no promoter activity of their own but they greatly increase the activities of many promoters in eukaryotes.

• Enhancers function by serving as binding sites for specific regulatory proteins.

• An enhancer is effective only in the specific cell types in which appropriate regulatory proteins are expressed.

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• Enhancer elements can exert their positive influence on transcription even when separated by thousands of base pairs from a promoter;

• they work when oriented in either direction; and they can work upstream (5') or downstream (3') from the promoter.

• Enhancers are promiscuous (indiscriminate); they can stimulate any promoter in the vicinity and may act on more than one promoter.

• Promoters are capable of initiating lower levels of transcription

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Binding of other transcription factors, cofactors; RNA polymerase etc.

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• The elements that decrease or repress the expression of specific genes have also been identified.

• Silencers are control regions of DNA that, like enhancers, may be located thousands of base pairs away from the gene they control.

• However, when transcription factors bind to them, expression of the gene they control is repressed.

• Tissue-specific gene expression is mediated by enhancers or enhancer-like elements.

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Down Stream Promoter element

Gene Rearrangement

• Gene rearrangement is observed during immunoglobulins synthesis.

• Immunoglobulins are composed of two polypeptides, heavy (about 50 kDa) and light (about 25 kDa) chains.

• The mRNAs encoding these two protein subunits are encoded by gene sequences that are subjected to extensive DNA sequence-coding changes.

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These DNA coding changes are needed for generating the required recognition diversity central to appropriate immune function.

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• The IgG light chain is composed of variable (VL), joining (JL), and constant (CL) domains or segments.

• For particular subsets of IgG light chains, there are roughly 300 tandemly repeated VL gene coding segments, five tandemly arranged JL coding sequences, and roughly ten CL gene coding segments.

• All of these multiple, distinct coding regions are located in the same region of the same chromosome.

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• However, a given functional IgG light chain transcription unit contains only the coding sequences for a single protein.

• Thus, before a particular IgG light chain can be expressed, single VL, JL, and CL coding sequences must be recombined to generate a single, contiguous transcription unit excluding the multiple non utilized segments.

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• This deletion of unused genetic information is accomplished by selective DNA recombination that removes the unwanted coding DNA while retaining the required coding sequences: one VL, one JL, and one CL sequence.

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Alternative RNA Processing

• Eukaryotic cells also employ alternative RNA

processing to control gene expression.

• This can result when alternative promoters, intron-exon splice sites, or polyadenylation sites are used.

• Occasionally, heterogeneity within a cell results, but more commonly the same primary transcript is processed differently in different tissues.

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• Alternative polyadenylation sites in the immunoglobulin (Ig M) heavy chain primary transcript result in mRNAs that are either 2700 bases long (m) or 2400 bases long (s).

• This results in a different carboxyl terminal region of the encoded proteins such that the (m ) protein remains attached to the membrane of the B lymphocyte and the (s) immunoglobulin is secreted.

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Alternative RNA Processing (contd.)

Alternative splicing and processing, results in the formation of seven unique -tropomyosin mRNAs in seven different tissues.

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Class switching

In this process one gene is switched off and a closely related gene takes up the function.

During intrauterine life embryonic Hb is the first Hb to be formed.

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• It is produced by having two “Zeta” and two “Epsilon” chains.

• By the sixth month of intrauterine life, embryonic Hb is replaced by HbF consisting of “α2 and y2 chains.

• After birth HbF is replaced by adult type of

Hb A1(97%) and HbA2(3%).

• Thus the genes for a particular class of Hb are switched off and for another class are switched on.

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Class switching (contd.)

• Gene switching is also observed in the formation of immunoglobulins.

• Ig M is the formed during primary immune response, while Ig G is formed during secondary immune response.

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mRNA stability

• Although most mRNAs in mammalian cells are very stable (half-lives measured in hours), some turn over very rapidly (half-lives of 10–30 minutes).

• In certain instances, mRNA stability is subject to regulation.

• This has important implications since there is usually a direct relationship between mRNA amount and the translation of that mRNA into its cognate protein ( enzyme and its normal substrate or a receptor and its normal ligand ).

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• Changes in the stability of a specific mRNA can therefore have major effects on biologic processes.

• The stability of the m RNA can be influenced by hormones and certain other effectors.

• The ends of mRNA molecules are involved in mRNA stability.

• The 5' cap structure in eukaryotic mRNA prevents attack by 5' exonucleases, and the poly(A) tail prohibits the action of 3' exonucleases.

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DNA binding proteins

• Steroids such as estrogens bind to eukaryotic transcription factors called nuclear hormone receptors. These proteins are capable of binding DNA whether or not ligands are bound.

• The binding of ligands induces a conformational change that allows the recruitment of additional proteins called

co-activators. 36

• Among the most important functions of co-activators is catalysis of the addition of acetyl groups to lysine residues in the tails of histone proteins.

• Histone acetylation decreases the affinity of the histones for DNA, making additional genes accessible for transcription.

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RNA Editing

• Enzyme- catalyzed deamination of a specific cytidine residue in the mRNA of apolipoprotein B-100 changes a codon for glutamine (CAA) to a stop codon (UAA).

• Apolipoprotein B-48, a truncated version of the protein lacking the LDL receptor-binding domain, is generated by this posttranscriptional change in the mRNA sequence.

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RNA Editing (contd.)

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Summary

• The genetic constitutions of nearly all metazoan somatic cells are identical.

• Tissue or cell specificity is dictated by differences in gene expression of this complement of genes.

• Alterations in gene expression allow a cell to adapt to environmental changes.

• Gene expression can be controlled at multiple levels by chromatin modifications ,changes in transcription, RNA processing, localization, and stability or utilization.

• Gene amplification and rearrangements also influence gene expression.

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Purpose of regulation of gene expression

Regulated expression of genes is required for

1) Adaptation- Cells of multicellular organisms respond to varying conditions.

Such cells exposed to hormones and growth factors change substantially in –

o shape,

o growth rate, and

o other characteristics

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Purpose of regulation of gene expression

2) Tissue specific differentiation and development The genetic information present in each somatic cell

of a metazoan organism (multicellular) is practically identical.

Cells from muscle and nerve tissue show strikingly different morphologies and other properties, yet they contain exactly the same DNA.

These diverse properties are the result of differences in gene expression.

Expression of the genetic information is regulated during development and differentiation of the organism and its cellular components.

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• END OF GENE REGULATION

Part III