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For gene expression, prokaryotes require one kind of RNA polymerase, in addition to three regulatory elements: promoter sequences, termination sequences, and operator sequences.
Eukaryotes require three RNA polymerases:
* RNA polymerase I synthesizes ribosomal RNA (rRNA is 90% of RNA in cell) and needs accessory factors to initiate transcription (positive control factors). RNA polymerase I appears to be species specific because rDNA from one species often can not be transcribed by the RNA polymerase I from another species.
* RNA polymerase II synthesizes pre-messenger RNAs (pre-mRNAs). It is the most delicately regulated eukaryotic RNA polymerase, it needs accessory proteins (transcription factors) and is not species specific.
* RNA polymerase III is responsible for the synthesis of tRNA, 5S RNA and snRNA (small nuclear RNA).
Transcription Termination
For RNA polymerase I and usually for III, there exists a specific termination signal (sequence of nucleotides) on the DNA. This sequence tells the RNA polymerase when to stop the transcription process and release the RNA.
For RNA polymerase II, however, there is no specific termination sequence. The RNA polymerase continues until it "falls off," then the precursor RNA is trimmed at a specific site to produce the final pre-mRNA product.
Since the RNA is produced in the nucleus and is necessary for protein synthesis in the cytoplasm, the RNA must be transported out of the nucleus. This process is controlled by a regulating system.
Characteristics of pre-mRNAs
When the pre-mRNA is synthesized a cap is added to the 5' end of the pre-mRNA. This specific unique structure interacts with eukaryotic ribosomes to help initiate translation. The cap serves as a binding site for ribosomes in the translation process.
Since the cap is needed for initiation of translation, a polycistronic eukaryotic mRNA will not function because the cap can only reside at the 5' end of the RNA - the middle cistrons cannot be translated because they do not have a nearby cap. Thus, there are no polycistronic RNAs in eukaryotes, only in prokaryotes.
The 3' end is formed when the pre-mRNA recognizes a sequence downstream of the 3' end of the coding region (called the 3' untranslated region, or the trailer sequence): AAUAAA. This sequence specifies that the pre-mRNA is to get cut at about 20 nucleotides past the AAUAAA sequence. A string of adenosines called the poly(A) tail is then added to the 3' end of pre-mRNA. The poly(A) tail seems to protect the RNA from degradation in the cell. Without it, the mRNA would be degraded in a matter of minutes. So the poly(A) tail serves to help stabilize the mRNA. The length of the poly(A) tail may be related to the age of that mRNA strand.
Eukaryotic pre-mRNAs also contain introns (for intervening sequences), which are sequences between coding sequences (called exons for expressed sequences) that don't code for anything. The process of RNA splicing removes introns and joins the exons together to create mature mRNAs that consist only of a usable sequence, and are ready for export from nucleus to cytoplasm.
Methylation of DNA
Methyl groups (-CH3) attach themselves to the cytosines that reside in CG (cytosine-guanine) doublets on DNA. Extensive methylation near a gene promoter on DNA will generally prevent transcription of that gene. If the methyl groups are removed, transcription can, but not necessarily will, occur again. In other words, the absence of methylation is a necessary but not sufficient condition for transcription to occur.
Enzymes that add methyl groups are called methylases - they recognize certain locations on DNA and add methylation to them.
No demethylase enzymes have been found; that is, no enzyme has been discovered that can remove the methyl groups once they become attached to the DNA. The only way methyl groups can be removed is through the creation of new DNA strands, which are not methylated at first - this only occurs during DNA replication.
If a demethylase could be found and inserted into cells, it might allow rejuvenation of cells, but there could be consequences to demethylization: preventing methylation could turn genes on and off, could create tumors and other problems. Methylation cannot be a universal method of regulation gene expression because at least some organisms (like fruit flies) don't methylate their DNA.
Transcription of Eukaryotic Genes/Transcription Factors
Before a gene can be turned on for transcription, it must be physically exposed to interact with control factors. Since DNA is packed into chromatin which is organized into nucleosomes, the chromatin must first unravel so as to make the binding sites on the DNA available.
Proteins called transcription factors regulate the level of transcription from a given gene. These transcription factors are similar to gene activators in prokaryotic cells. Each transcription factor recognizes a specific sequence on DNA. Since the transcription factors can also bind to DNA sequences which are close to (but not identical to) the ideal sequence, the binding sequence is often called a consensus sequence. This means that certain DNA sequences could be recognized by more than one transcription factor if the sequences are similar to those recognized by two different transcription factors.
Each element of the promoter might be capable of recognizing a few different transcription factors and each transcription factor is capable of recognizing a number of promoter elements. If more than one transcription factor can bind to a particular promoter element, the different transcription factors will compete for binding to that element. The level of transcription from the regulated gene will depend on the number of different preceding transcription factors.
Enhancers
Some sequences don't seem to be connected with the promoter of a gene but still can influence the level of transcription from that gene. These elements, called enhancers, exist elsewhere on the DNA but must reside on the same DNA molecule to work. Enhancers, which can point in either direction, can be up to 20,000 basepairs away from the gene and can be present upstream, downstream, or even internally with respect to the gene it is regulating. Enhancers can increase the level of transcription by stimulating transcription from all nearby genes. Some transcription factors can also recognize and bind to enhancer elements. This allows for additional control on broader level, providing control of a set of connected genes.
Problems can arise if enhancers are where they shouldn't be, for example: viruses can have enhancers in their genome that when inserted near a gene, turn on a promoter and express a gene that wasn't being expressed previously.
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