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Lecture NO 1: Gene Expression

In genetics gene expression is the most fundamental level at which genotype gives rise to the phenotype. The genetic code is "interpreted" by gene expression, and the properties of the expression products give rise to the organism's phenotype.

Steps of Gene expression:

Transcription:
Transcription is the production of RNA copies of the DNA which is performed by RNA polymerase. Worth mentioning note: This RNA is complementary to the DNA nucleotide being transcribed; i.e. a T on the DNA means an A is added to the RNA. However, in RNA the nitrogen-containing base Uracil is inserted instead of Thymine wherever there is an Adenine on the DNA strand. Therefore, the mRNA complement of a DNA
strand reading "TAC" would be transcribed as "AUG".

RNA processing:
The most common RNA processing is splicing to remove
introns. Introns are RNA segments which are not found in the mature RNA, although they can function as precursors, e.g. for snoRNAs, which are RNAs that direct modification of nucleotides in other RNAs. Introns are common in eukaryotic genes but rare in prokaryotes.

RNA export
In eukaryotes most mature RNA must be exported to the
cytoplasm from the nucleus. While some RNAs function in the nucleus, many RNAs
are transported through the nuclear pores and into the cytosol. Notably this
includes all RNA types involved in protein synthesis.

Translation
For some RNA (non-coding RNA) the mature RNA is the finished gene product. In the case of messenger RNA (mRNA) the RNA is an information carrier coding for the synthesis of one or more proteins. mRNA carrying a single protein sequence (common in eukaryotes) is monocistronic whilst mRNA carrying multiple protein sequences (common in prokaryotes) is known as polycistronic. Each triplet of nucleotides of the coding regions 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 to bind to messenger RNA and takes the amino acid from each transfer RNA and makes a structure-less protein out of it. In prokaryotes translation generally occurs at the point of
transcription, often using a messenger RNA which is still in the process of being created. In eukaryotes translation can occur in a variety of regions of the cell depending on where the protein being written is supposed to be. Major locations are the cytoplasm for soluble cytoplasmic proteins and the endoplasmic reticulum for proteins which are for export from the cell or insertion into a cell membrane. Proteins which are supposed to be expressed at the endoplasmic reticulum are recognised part-way through the translation process. This is governed by the signal recognition particle - a protein which
binds to the ribosome and directs it to the endoplasmic reticulum when it finds a signal sequence on the growing (nascent) amino acid chain. Enzymes called chaperones assist the newly formed protein to attain (fold into) the 3-dimensional structure it needs to function. Similarly, RNA
chaperones help RNAs attain their functional shapes. Assisting protein folding is one of the main roles of the endoplasmic reticulum in eukaryotes.

Protein transport
Many proteins are destined for other parts of the cell than
the cytosol and a wide range of signalling sequences are used to direct proteins to where they are supposed to be. In prokaryotes this is normally a simple process due to limited compartmentalisation of the cell. However in eukaryotes there is a great variety of different targeting processes to ensure
the protein arrives at the correct organelle. Not all proteins remain within the cell and many are exported, for example digestive enzymes, hormones and extracellular matrix proteins. In eukaryotes the export pathway is well developed and the main mechanism for the export of these proteins is translocation to the endoplasmatic reticulum, followed by transport via the Golgi apparatus.


Gene regulation
Several steps in the gene expression process may be
modulated, including the transcription, RNA splicing, translation, and 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.

Definition and importance:

Regulation of gene expression refers to the control of the amount and timing of appearance of the functional product of a gene. Control of expression is vital to allow a cell to produce the gene products it needs when it needs them.

Examples: Control of Insulin expression so it gives a signal for blood glucose regulation Cyclin expression levels control progression through the eukaryotic cell cycle. Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein. The stability of the final gene product, whether it is RNA or protein, also contributes to the expression level
of the gene - an unstable product results in a low expression level. In general gene expression is regulated through changes in the number and type of interactions between molecules that collectively influence transcription of DNA and translation of RNA.

Transcriptional regulation
Regulation of transcription can be broken down into three
main routes of influence; genetic (direct interaction of a control factor with the gene), modulation (interaction of a control factor with the transcription machinery) and epigenetic (non-sequence changes in DNA structure which influence transcription). Direct interaction with DNA is the simplest and most direct method a protein can change transcription levels and genes often have several protein binding sites around the coding region with the specific function of regulating transcription. There are many classes of regulatory DNA binding sites known as enhancers, insulators, repressors and silencers. The mechanisms for regulating transcription are very varied, from blocking key binding sites on the DNA for RNA
polymerase to acting as an activator and promoting transcription by assisting RNA polymerase binding.
The activity of transcription factors is further modulated
by intracellular signals causing protein post-translational modification including phosphorylated, acetylated, or glycosylated. These changes influence a transcription factor's ability to bind, directly or indirectly, to promoter DNA, to recruit RNA polymerase, or to favor elongation of a newly synthetized RNA molecule. More recently it has become apparent that there is a huge influence of non-DNA-sequence specific effects on translation. These effects are referred to as epigenetic and involve the higher order structure of DNA,
non-sequence specific DNA binding proteins and chemical modification of DNA. In general epigenetic effects alter the accessibility of DNA to proteins and so modulate
transcription.

DNA methylation:
Methylation of DNA (not to be confused with histone methylation) is a common epigenetic signaling tool that cells use to lock genes in the "off" position. DNA methylation is a widespread mechanism for epigenetic influence on gene expression and is seen in bacteria and eukaryotes and has roles in heritable transcription silencing and transcription regulation. In eukaryotes the structure of chromatin, controlled by the histone code, regulates access to DNA with significant impacts on the expression of genes in euchromatin and heterochromatin areas.

Post-Transcriptional regulation
In eukaryotes, where export of RNA is required before
translation is possible, nuclear export is thought to provide additional control over gene expression. All transport in and out of the nucleus is via the nuclear pore and transport is controlled by a wide range of importin and exportin proteins.
RNA stability Expression of a gene coding for a protein is only possible if the messenger RNA carrying the code survives long enough to be translated. In a typical cell an RNA molecule is only stable if specifically protected from degradation. RNA
degradation has particular importance in regulation of expression in eukaryotic cells where mRNA has to travel significant distances before being translated. In eukaryotes RNA is stabilised by certain post-transcriptional modifications,
particularly the 5' cap and poly-adenylated tail. Intentional degradation of mRNA is used not just as a defence mechanism from foreign RNA (normally from viruses) but also as a route of mRNA ''destabilisation''. If an mRNA molecule has a
complementary sequence to a small interfering RNA then it is targeted for destruction via the RNA interference pathway.
Translational regulation Direct regulation of translation is less prevalent than control of transcription or mRNA stability but is occasionally used. Inhibition of protein translation is a major target for toxins and antibiotics in order to kill a cell by overriding its normal gene expression control. Protein synthesis inhibitors include the antibiotic neomycin and the toxin ricin.

Protein degradation and turnover
Once protein synthesis is complete the level of expression
of that protein can be reduced by protein degradation. There are major protein degradation pathways in all prokaryotes and eukaryotes of which the proteasome is a common component. An unneeded or damaged protein is often labelled for
degradation by addition of ubiquitin.