Gene Expression

Gene expression is the process by which the inheritable information in a gene, such as the DNA sequence, is made into a functional gene product, such as protein or RNA.

Several steps in the gene expression process may be modulated, including the transcription step and the post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene in the organism.

Non-protein coding genes (e.g. rRNA genes, tRNA genes) are not translated into proteins.

Measurement

The expression of many genes is regulated after transcription (i.e., by microRNAs or ubiquitin ligases), so an increase in mRNA concentration need not always increase expression. Nevertheless, mRNA levels can be quantitatively measured by Northern blotting, a process in which a sample of RNA is separated on an agarose gel and hybridized to a radio-labeled RNA probe that is complementary to the target sequence. Northern blotting requires the use of radioactive reagents and can have lower data quality than more modern methods (due to the fact that quantification is done by measuring band strength in an image of a gel), but it is still often used. It does, for example, offer the benefit of allowing the discrimination of alternately spliced transcripts.

A more modern low-throughput approach for measuring mRNA abundance is real-time polymerase chain reaction or RT-PCR. With a carefully constructed standard curve RT-PCR can produce an absolute measurement such as number of copies of mRNA per nanolitre of homogenized tissue. The lower level of noise in data obtained via RT-PCR often makes this the method of choice, but the price of the required equipment and reagents can be prohibitive.

In addition to low-throughput methods, transcript levels for many genes at once (expression profiling) can be measured with DNA microarray technology or "tag based" technologies like Serial analysis of gene expression (SAGE) or the more advanced version SuperSAGE, which can provide a relative measure of the cellular concentration of different messenger RNAs. Recent advances in microarray technology allow for the quantification, on a single array, of transcript levels for every known gene in the human genome. The great advantage of tag-based methods is the "open architecture", allowing for the exact measurement of any transcript, known or unknown. Especially SuperSAGE recommends itself therefore also for studying organisms with unknown genomes.

Protein levels themselves can be estimated by a number of means. The most commonly used method is to perform a Western blot against the protein of interest, whereby cellular lysate is separated on a polyacrylamide gel and then probed with an antibody to the protein of interest. The antibody can either be conjugated to a fluorophore or to horseradish peroxidase for imaging or quantification. Another commonly used method for assaying the amount of a particular protein in a cell is to fuse a copy of the protein to a reporter gene such as Green fluorescent protein, which can be directly imaged using a fluorescent microscope. Because it is very difficult to clone a GFP-fused protein into its native location in the genome, however, this method often cannot be used to measure endogenous regulatory mechanisms (GFP-fusions are therefore most often expressed on extra-genomic DNA such as an expression vector). Fusing a target protein to a reporter can also change the protein's behavior, including its cellular localization and expression level.

The pattern of detection of a gene or gene product may be described using terms such as facultative, constitutive, circadian, cyclic, housekeeping, or inducible.

Regulation of gene expression

Regulation of gene expression is the cellular control of the amount and timing of appearance of the functional product of a gene. Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism.

Expression system

An expression system consists, minimally, of a source of DNA and the molecular machinery required to transcribe the DNA into mRNA and translate the mRNA into protein using the nutrients and fuel provided. In the broadest sense, this includes every living cell capable of producing protein from DNA. However, an expression system more specifically refers to a laboratory tool, often artificial in some manner, used for assembling the product of a specific gene or genes. It is defined as the "combination of an expression vector, its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level".

In addition to these biological tools, certain naturally observed configurations of DNA (genes, promoters, enhancers, repressors) and the associated machinery itself are referred to as an expression system, as in the simple repressor 'switch' expression system in Lambda phage. It is these natural expression systems that inspire artificial expression systems, (such as the Tet-on and Tet-off expression systems).

Each expression system has distinct advantages and liabilities, and may be named after the host, the DNA source or the delivery mechanism for the genetic material. For example, common expression systems include bacteria (such as E.coli), yeast (such as S.cerevisiae), plasmid, artificial chromosomes, phage (such as lambda), cell lines, or virus (such as baculovirus, retrovirus, adenovirus).

Overexpression

In the laboratory, the protein encoded by a gene is sometimes expressed in increased quantity. This can come about by increasing the number of copies of the gene or increasing the binding strength of the promoter region.

Often, the DNA sequence for a protein of interest will be cloned or subcloned into a plasmid containing the lac promoter, which is then transformed into the bacterium Escherichia coli. Addition of IPTG (a lactose analog) causes the bacteria to express the protein of interest. However, this strategy does not always yield functional protein, in which case, other organisms or tissue cultures may be more effective. As for example the yeast, Saccharomyces cerevisiae, is often preferred to bacteria for proteins that undergo extensive Posttranslational modification. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination.

Gene networks and expression

Genes have sometimes been regarded as nodes in a network, with inputs being proteins such as transcription factors, and outputs being the level of gene expression. The node itself performs a function, and the operation of these functions have been interpreted as performing a kind of information processing within cell and determine cellular behaviour.

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Histones Animation

Histones are the chief protein components of chromatin. They act as spools around which DNA winds and they play a role in gene regulation.

Classes

Six major histone classes are known:
H1 (sometimes called the linker histone or H5.)
H2A
H2B
H3
H4
Archaeal histones

Two each of the class H2A, H2B, H3 and H4, so-called core histones, assemble to form one octameric nucleosome core particle by wrapping 146 base pairs of DNA around the protein spool in 1.65 left-handed super-helical turn. The linker histone H1 binds the nucleosome and the entry and exit sites of the DNA, thus locking the DNA into place and allowing the formation of higher order structure. The most basic such formation is the 10 nm fiber or beads on a string conformation. This involves the wrapping of DNA around nucleosomes with approximately 50 base pairs of DNA spaced between each nucleosome (also referred to as linker DNA). Higher order structures include the 30 nm fiber (forming an irregular zigzag) and 100 nm fiber, these being the structures found in normal cells. During meiosis, through the combination of nucleosome interactions with other proteins, the chromosome is assembled. The assembled histones and DNA is called chromatin.

Two each of the class H2A, H2B, H3 and H4, so-called core histones, assemble to form one octameric nucleosome core particle by wrapping 146 base pairs of DNA around the protein spool in 1.65 left-handed super-helical turn. The linker histone H1 binds the nucleosome and the entry and exit sites of the DNA, thus locking the DNA into place and allowing the formation of higher order structure. The most basic such formation is the 10 nm fiber or beads on a string conformation. This involves the wrapping of DNA around nucleosomes with approximately 50 base pairs of DNA spaced between each nucleosome (also referred to as linker DNA). Higher order structures include the 30 nm fiber (forming an irregular zigzag) and 100 nm fiber, these being the structures found in normal cells. During meiosis, through the combination of nucleosome interactions with other proteins, the chromosome is assembled. The assembled histones and DNA is called chromatin.

Structure

The nucleosome core is formed of two H2A-H2B dimers and a H3-H4 tetramer, forming two nearly symmetrical halves by tertiary structure (C2 symmetry; one macromolecule is the mirror image of the other). The H2A-H2B dimers and H3-H4 tetramer also show pseudodyad symmetry.

The 4 'core' histones (H2A, H2B, H3 and H4) are relatively similar in structure and are highly conserved through evolution, all featuring a 'helix turn helix turn helix' motif (which allows the easy dimerisation). They also share the feature of long 'tails' on one end of the amino acid structure - this being the location of post-transcriptional modification

In all, histones make five types of interactions with DNA:
Helix-dipoles from alpha-helices in H2B, H3, and H4 cause a net positive charge to accumulate at the point of interaction with negatively charged phosphate groups on DNA.
Hydrogen bonds between the DNA backbone and the amine group on the main chain of histone proteins.
Nonpolar interactions between the histone and deoxyribose sugars on DNA.
Salt links and hydrogen bonds between side chains of basic amino acids (especially lysine and arginine) and phosphate oxygens on DNA.
Non-specific minor groove insertions of the H3 and H2B N-terminal tails into two minor grooves each on the DNA molecule.

The highly basic nature of histones, aside from facilitating DNA-histone interactions, contributes to the water solubility of histones.

Histones are subject to posttranslational modification by enzymes primarily on their N-terminal tails, but also in their globular domains. Such modifications include methylation, citrullination, acetylation, phosphorylation, Sumoylation, ubiquitination, and ADP-ribosylation. This affects their function of gene regulation

In general, genes that are active have less bound histone, while inactive genes are highly associated with histones during interphase. It also appears that the structure of histones have been evolutionarily conserved, as any deleterious mutations would be severely maladaptive.

Functions

Packing proteins
Histones act as spools around which DNA winds. This enables the compaction necessary to fit the large genomes of eukaryotes inside cell nuclei: the compacted molecule is 50,000 times shorter than an unpacked molecule.

Histone modifications in chromatin regulation

Histones undergo posttranslational modifications which alter their interaction with DNA and nuclear proteins. The H3 and H4 histones have long tails protruding from the nucleosome which can be covalently modified at several places. Modifications of the tail include methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination, and ADP ribosylation. The core of the histones (H2A and H3) can also be modified. Combinations of modifications are thought to constitute a code, the so-called "histone code". Histone modifications act in diverse biological processes such as gene regulation, DNA repair and chromosome condensation (mitosis)

Secretory Pathway Animation

The secretory pathway is a series of steps a cell uses to move proteins out of the cell; a process known as secretion. The path of a protein destined for secretion has its origins in the rough endoplasmic reticulum, a membrane bound compartment in the cell. The protein then proceeds through the many compartments of the Golgi apparatus and finally ends up in a vesicle that fuses with the plasma membrane, dumping the proteins outside of the cell. At each step along the way there are crucial factors that determine how and if the protein will proceed. Some of these factors include regulation of transportation, selection of particular proteins, the mechanics of proceeding to the next step, and modifications that can occur to the protein along the way. All of these factors contribute to how a protein arrives outside of a cell after being synthesized.

General Regulation

In general, there are two different patterns of secretion. One pattern is called constitutive secretion. Proteins are continuously secreted from the cell regardless of environmental factors. No external signals are needed to initiate this process. Proteins are packaged in vesicles in the Golgi apparatus and are secreted via exocytosis, all around the cell. Cells that secrete constitutively have many Golgi apparatus scattered throughout the cytoplasm. Fibroblasts, osteoblasts and chondrocytes are some of the many cells that perform constitutive secretion.

In regulated secretion, proteins are packaged as described in the constitutive pathway, but they are only secreted in response to a specific signal, such as neural or hormonal stimulation. Cells that use the regulated secretory pathway are usually apical or polarized. The Golgi apparatus are found in a supranuclear position (between the nucleus and the secretory surface). Examples of cells that use regulated pathway are: goblet cells (secrete mucus), beta cells of the pancreas (secrete insulin) and odontoblasts (secrete dentin).

Protein Translocation

The first step in a protein's journey out of the cell is getting into the endoplasmic reticulum. Two methods exist for proteins to accomplish this. One is co-translational translocation and the other is post-translational translocation.

Co-Translational translocation is the action of a protein being fed into the ER as it is synthesized by the ribosome. In this scenario the protein begins to be translated normally. As the N-terminus (the first end of the protein to be synthesized) leaves the ribosome, a short sequence of the nascent protein's amino acids is recognized by a protein complex called SRP, or signal recognition particle. SRP binds to this sequence of amino acids and then subsequently binds to a protein complex embedded in the ER membrane called the SRP receptor. Both the SRP complex and SRP receptor complex hydrolyze a molecule of GTP into GDP. Once this occurs the growing protein chain is moved to a membrane channel called the translocon. It is through this hole in the ER membrane that the protein passes through as it is synthesized. The result of all this is a new protein residing within the membrane of the endoplasmic reticulum.

Post-Translational translocation is the action of a protein passing into the ER after being fully synthesized by the ribosome.

Gene Regulation Lecture