Programs and Regulatory Elements in DNA and RNA expression

This page is dedicated to the second learning objective pretaining to the regulatory elements discussed in class.

Exons and Introns
One of the major differences between the prokaryotic and eukaryotic genomes is that eukaryotic genes are not co-linear. The single nucleic acid strand of eukaryotic mRNA comes from non-continuous regions on the chromosome.

Between exons- the regions of DNA that code for a protein- are intervening sequences called introns that have nothing to do with the amino acid sequence of the protein.

Most genes consist of the following components:

[[File:B-Globin.png|thumb|400px|Summary of steps involved in the production of B-Globin and Hemoglobin.

Development Biology, 9e, Gilbert p39.]]


 * Promoter region: responsible for the binding of RNA polymerase
 * Transcriptional initiation site
 * This site is often caled the cap sequence becuase it represents the 5' end of the RNA
 * Translation initiation site (ATG)
 * 5' Untranslated Region (UTR)
 * Translation termination codon
 * 3' UTR
 * Transcription termination sequence

Hemoglobin Example
The following figure depicts the summary of steps that are involved in the production of hemoglobin.

Transcription of the gene creates a nuclear RNA containing exons and introns, as well as the cap, tail, and 3' and 5' untranslated regions. Processing the nuclear RNA into messenger RNA removes the inrons. Translation on ribosomes uses the mRNA to encode a protein. The protein is inactive until it is modified and complexed with a-globin and heme to become the active form of Hemoglobin.

Promoters
Promoters and Enhancers are regulatory sequences that are necessary for the control of when and where a particular gene is transcribed.

Promoters are the sites where RNA polymerase binds to the DNA to initiate transcription. They are usually located immediately upstream from the gene. Most promoters contain a TATA Box. Since 'TATA' can appear randomly throughout the genome, important TATA boxes are usually flanked by CpG islands (important later in epigenetic control).

RNA polymerase will not bind to the naked TATA box; they require the presence of additional proteins to place the polymerase properly on the promoter. TATA Binding proteins (TBP) form complexes with Transcription factors A-H to form the 'Basal Transcription Machinery'.

Enhancers and Transcription factors
An enhancer is a DNA sequence that controls the efficiency and rate of transciption from a specific promoter. Enhancers bind to specific transcription factors that activate the gene by (1) recruiting enzymes  that interact with the nucleosome and (2) stablizing the transcription initiation complex.

Enhancers can only activate cis-linked promoters (ie promoters on the same chromosome) and thats why enhancers can also be refered to as cis-regulatory elements.

Reporter Genes
One way to identify the activity of an enhancer is to clone the enhancer sequence and flank them to a Reporter gene. Reporter genes produce a product that is not endogenous to the animal system and has an identifiable characteristic.

One reporter gene that is commonly used in animal systems is the LacZ gene. The LacZ gene encodes for B-Galactosidase which can be easily stained and viewed in the developing organism.

Example of Reporter gene use:


 * Let us imagine that there exists a gene of interest that has a high activity in the development of the spinal cord in a mouse. We identify the sequence and we then are interested when and where the gene is expressed. It is most likely that there are enhancers that control gene expression. One way to identify the enhancers is thrugh the use of reporter genes. We genetically engineer an upstream portion of the gene of interest and fuse it to the LacZ reporter gene. We then introduce the construct back into the mouse system and stain for B-Galactosidase. If a enhancer is caught in the contruct, we should see a blue staining at certain times and locations in the development of the spinal cord. The blue stain is the result of the report gene being activated by the enhancers that were caught.

Enhancer Modularity
Enhancers are the same in every cell type, what differs is the transcription factors that associate with the enhancers.

It is the combination of specific transcription factors with enhancers that express genes at different times and locations.

Example: PAX6 gene
 * The Pax6 gene is expressed in both the eye and the pancreas and has several different enhancers
 * Four main enhancers where found to correspond to the Pax6 gene. The first, and fartherest upstream, was found to express Pax6 in the pancreas. The fourth enhancer was found to exist in the intron of the PAX6 gene and expressed the gene in the retina.

Differential RNA Processing
The next stage of gene expression is done at the RNA level. The two major ways that genes are controlled at the RNA level is through (1) Censorship of nRNA and (2) Alternative splicing.

nRNA censoring
Genes are constantly being transcribed in the nucleus that are not needed to be translated in the cytoplasm. A way of control and a method of cell specialization is through the 'censoring' of which nRNA messages are able to leave the nucleus to be translated into protein.

Example: CyIIIa gene in Sea Urchins


 * The CyIIIa gene encode for actin proteins in the Sea Urchin whihc are only expressed in a particular part of the ectoderm. Using intro/exon probes, it was found that the CyIIIa gene was being transcribed in all the cells of the sea urchin but only translated in the ectoderm. The nRNA product of CyIIIa was quickly degraded in the nucleus before it was allowed to enter the cytoplasm.

Alternative Splicing
It was noted that the total genomic content differed among different species. For instance, Humans and nematodes share a similar total genomic content while some plants have a larger total genomic content. Paradoxically, the size of the genome did not correlate with the complexity of the organism. This observation is due to the activity of alternative splicing.

Alternative splicing allows for differential splicing and processing of an RNA product to produce multiple proteins. In humans, RNA can be spliced in many different ways to form a family of different protein isoforms (aka Splicing Isoforms). Genes that produce multiple proteins contain splice sites that are recgonized by spliceosomes and other splicing machinery to excise certain exons and assemble the final mRNA product to be translated.



Example: a-tropomyosin in rats


 * A great example of alternative splicing comes from the a-tropomyosin gene in rats. It has been found that there are 7 major protein isoforms that come from this single gene. Each protein isoform has a specific function in different specialized tissue. For instance, one protein is translated specifically for the brain while another is translated specifically for smooth muscle.


 * Depending on how the exons are spliced and put together determines which specific protein is translated. For instance, the mRNA that encodes for the brain specific protein is missing the 3' UTR that is found in the pre-spliced mRNA while the mRNA that encodes for smooth muscle specific protein contains the 3' UTR,

Control of Gene expression at the Translational level
The next level of control in gene expression is at the level of protein translation. The are three major ways that control is done at the translational level: (1) mRNA longevity, (2) microRNA and (3) localization.

mRNA longevity
The longer that a mRNA is present in the cytoplasm, the more protein can be translated from it. The longevity of a mRNA is determined by its overall stability in the cytoplasm. Overall stability of the mRNA is governed by the length of its poly A tail modification. Overall, the longer the poly-A-tail, the longer lifespan the mRNA will have in the cytoplasm. Depending on the cell type or situation, a mRNA can be selectively stablized at specific times.

Example: Casein in Rat Mammary Gland.


 * Casein, the main protien found in milk, has a very short half-life in Rat mammary tissues. However, in a time of lactation, the presences of certain horomones act to increase the life-span of casein significantly.

microRNA
microRNA are hairpin RNA strands that interact with specific proteins to slience the production of their downstream mRNA targets. The microRNA interacts with a protein named DICER to be unwound into a single RNA strand. This single RNA strand is then complexed with the RISC complex with it allows the microRNA strand to bind to its target mRNA. Targeted mRNA is then cleaved and ultimately destroyed by RISC.

microRNA is usually used to adjust the levels of mRNA product that exists in the cell.



Cytoplasmic Localization
mRNA translation is controlled both in time and in space. Localization of mRNA can lead to translation at different physical location in cells and embryos (as we will see with Drosophila). Localization of mRNA is accomplished by three mechanisms:

1) Diffusion and local anchoring (ex nanos)


 * The mRNA product flows freely throughout the cell in an inactive state. When the mRNA floats to a specific region it may be trapped by proteins that reside there. When the mRNA is complexed with those proteins they are translated.

2) Localization Protection (Drosophila Heatshock proteins)


 * Similarily to local anchoring, the mRNA's float freely throughout the cell to be caputred by proteins in specific regions. All mRNA that are not complexed with these proteins are actively degraded by Deadenylase complexes which significantly decrease the mRNA's half-life.

3) Active transport along the cytoskeleton (oskar and bicord)


 * Certain mRNA's are carried by transport proteins to their protein receptors. Ultimately, the region in which the mRNA is expressed is governed by the direction in which the transport protein carries the mRNA.



