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Gene expression

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For vocabulary, see Glossary of gene expression terms.
For a non-technical introduction to the topic, see Introduction to genetics.
Genes are expressed by being transcribed into RNA, and this transcript may then be translated into protein.

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA.

Several steps in the gene expression process may be modulated, including the transcription step and translation 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.

Mechanism

Transcription

The gene itself is typically a long stretch of DNA and does not perform an active role. It is a blueprint for the production of RNA. The production of RNA copies of the DNA is called transcription, and is performed by RNA polymerase, which adds one RNA nucleotide at a time to a growing RNA strand. This RNA is complementary to the DNA nucleotide being read, i.e. a T on the DNA means an A is added to the RNA.[1] However, in RNA the nitrogen-containing base Uracil is inserted instead of Thymine. Wherever there is an Adenine on the DNA strand, a Uracil is inserted into the complementary RNA strand. Therefore, the mRNA complement of a DNA strand reading "TAC" would be transcribed as "AUG", which is translated into the amino acid methionine, which is generally the starting point in a messenger RNA for expressing a protein.

RNA processing

Transcription creates a primary transcript of RNA at the place where the gene was located. This transcript often needs to be altered by enzymes. RNA processing, also known as post-transcriptional modification, can start already during transcription, as is the case for e.g. splicing where the spliceosome removes introns from newly formed parts of the RNA.[2] Introns are RNA segments which are not found in the mature RNA, although they can function as precursors for e.g. snoRNA which are a group of RNAs that direct nucleotide modification of other RNAs.[3]

In some cases large aggregates of RNA and RNA processing factors are formed, notably the nucleolus where ribosomal RNA localises to be processed by snoRNAs and their partner enzymes. These chop the primary ribosomal RNA transcripts into the correct segments and alter some of its nucleotides into e.g. pseudouridine.[4]

RNA export

While some RNAs function in the nucleus, many other RNAs in eukaryotes need to be transported through the nuclear pores and into the cytosol, including all the RNA types involved in protein synthesis.[5] In some cases the RNA is additionally transported to a specific part of the cytoplasm, such as a synapse, they are then towed by motor proteins that bind through linker proteins to specific sequences (called "zipcodes") on the RNA.[6]

Translation

For most RNA, the mature RNA is the gene product (see non-coding RNA).[7] In the case of messenger RNA however, the RNA is but an information carrier for the synthesis of a protein. Each triplet of nucleotides of the coding region of a messenger RNA corresponds to a binding site for a transfer RNA. Transfer RNAs carry amino acids, and these are chained together by the ribosome. The ribosome helps transfer RNA bind to messenger RNA and takes the amino acid from each tranfer RNA and makes a structure-less protein out of it.[8][9]

Some proteins have parts that should be within a membrane, these parts are moved into the membrane by the signal recognition particle which binds to the ribosome when it finds a signal sequence on the nascent amino acid chain.[10]

Folding

Enzymes called chaperones assist the newly formed protein to attain (fold into) the 3-dimensional structure it needs to function.[11] Similarly, RNA chaperones help RNAs attain their functional shapes.[12]

Protein export

Many proteins that are destined for other parts of the cell than the cytosol. A commonly used mechanism for transporting these proteins to where they should be is translocation to the endoplasmatic reticulum, followed by transport via the Golgi apparatus.[13][14]

Measurement

Levels of mRNA 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 (The term RT-PCR is used to refer to both reverse transcription PCR as well as real-time PCR, which is also known as quantitative RT-PCR or quantitative PCR (qPCR). With a carefully constructed standard curve qPCR 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 qPCR 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), 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.

The resultant protein levels 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.

Regulation of gene expression

Main article: 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".[15][16]

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, B. subtilis), 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. 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

Main article: Gene regulatory network

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.

Techniques and tools

The following experimental techniques are used to measure gene expression and are listed in roughly chronological order, starting with the older, more established technologies. They are divided into two groups based on their degree of multiplexity.

See also

References

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