RNA Polymerase
Transcription is the process whereby the directions for making a protein are converted from DNA-based instructions to RNA-based instructions. This step is required in the process of expressing a gene as a polypeptide, because ribosomes, which assemble polypeptides, can read only RNA-based messages. Although transcription is complicated and involves dozens of enzymes and proteins, it is much simpler in prokaryotes than in eukaryotes. Because prokaryotes
lack a nucleus, transcription and translation are linked processes both occurring in the cytoplasm. In eukaryotes, transcription and translation occur as completely separate processes, transcription occurring in the nucleus and translation occurring in the cytoplasm. (It is now known that some translation also occurs in the nucleus, but apparently only a small amount, probably less than 10 percent of the translation occurring in a cell.)
In eukaryotes there are three different types of RNA polymerase that transcribe RNA using a strand of DNA as a template (there is a single type of RNA polymerase in prokaryotes). Two of them, called RNA polymerase I (pol I) and RNA polymerase III (pol III), specialize in transcribing types of RNA that are functional products themselves, such as ribosomal RNA (rRNA) and transfer RNA (tRNA). These RNAs are involved in translation. RNA polymerase II (pol II) transcribes RNA from structural genes, that is, genes that code for polypeptides. Pol II therefore is the primary RNA polymerase and the one that will be the focus of this article when discussing transcription in eukaryotes.
Transcription in Prokaryotes
The first step in transcription is for RNA polymerase to identify the location of a gene. In prokaryotes many genes are clustered together in functional groups called operons. For example, the lactose (lac) operon contains three genes, each coding for one of the enzymes needed to metabolize the sugar lactose. At the beginning of each operon are two control sequences, the operator and the promoter. The promoter is where RNA polymerase binds, in preparation for transcription. The operator is a control region that determines whether RNA polymerase will be able to bind to the promoter. The operator interacts with other proteins that determine when the associated operon should be expressed. They do this by either preventing RNA polymerase from binding to the promoter or by assisting it to bind.
RNA polymerase recognizes promoters by the specific base-pair sequences they contain. Assuming all conditions are correct, RNA polymerase binds to the promoter, along with another protein called the sigma factor (σ). The beginning of genes are detected with the aid of σ. Transcription begins at a leader sequence a little before the beginning of the first gene and continues until RNA polymerase reaches a termination signal. If the operon contains more than one gene, all of the genes are transcribed into a single long mRNA, each gene separated from its neighbors by a spacer region. The mRNA is put together by pairing ribonucleotides with their complementary nucleotides in the DNA template. In place of thymine (T), RNA uses uracil (U); otherwise the same bases are present in RNA and DNA, the others being adenine (A), guanine (G), and cytosine (C). The pairing relationships are as follows, the DNA base listed first in each pair: A-U, T-A, G-C, and C-G.
RNA polymerase catalyzes the joining of ribonucleotides as they pair with the DNA template. Each mRNA is constructed beginning at the 5′ end (the phosphate end) and ending with the 3′ end (the hydroxyl end). Even while transcription is taking place, ribosomes begin binding to the mRNA to begin translation. As soon as RNA polymerase has completed transcribing the genes of an operon, it releases from the DNA and soon binds to another promoter to begin the process all over again.
Transcription in Eukaryotes
Transcription in eukaryotes differs from the process in prokaryotes in the following major ways: (1) Genes are transcribed individually instead of in groups; (2) DNA is complexed with many proteins and is highly compacted, and therefore must be “unwound” to expose its promoters; (3) transcription occurs in a separate compartment (the nucleus) from translation, most of which occurs in the cytoplasm; and (4) initially transcription results in a pre-messenger RNA (pre-mRNA) molecule that must be processed before it emerges as a mature mRNA ready for translation. Additionally, mRNAs are much longer-lived in eukaryotes.
The first step in transcription is for RNA polymerase to find a gene that needs to be transcribed. Only genes occurring in regions of the DNA that have been unwound are prepared for potential transcription. RNA polymerase binds to an available promoter, which is located just before a gene and has a region in it called the TATA box (all promoters have the consensus sequence TATAAAA in them). RNA polymerase is unable to bind to the promoter without assistance from more than a dozen other proteins, including a TATA-binding protein, several transcription factors, activators, and coactivators. There are other DNA sequences farther upstream than the promoter that control transcription too, thus accounting for the fact that some genes are transcribed more readily, and therefore more often, than others.
Once RNA polymerase has bound to the promoter, it begins assembling an RNA molecule complementary to the DNA code in the gene. It starts by making a short leader sequence, then transcribes the gene, and finishes after transcribing a short trailer sequence. Transcription ends when RNA polymerase reaches a termination signal in the DNA. The initial product is a pre-mRNA molecule that is much longer than the mature mRNA will be.
mRNA Processing in Eukaryotes
Pre-mRNAs must be processed before they can leave the nucleus and be translated at a ribosome. Three separate series of reactions play a part in producing a mature mRNA: (1) intron removal and exon splicing, (2) 5′ capping, and (3) addition of a poly-A tail. Not all transcripts require all three modifications, but most do.
The reason pre-mRNAs are much longer than their respective mature mRNAs has to do with the structure of genes in the DNA. The coding sequences of almost all eukaryotic genes are interrupted with noncoding regions. The noncoding regions are called introns, because they represent “intervening” sequences, and the coding regions are called exons. For an mRNA to be mature it must have all the introns removed and all the exons spliced together into one unbroken message. Special noncoding RNA complexes called small nuclear ribonucleoprotein particles, or snRNPs (pronounced as “snurps” by geneticists), carry out this process. The RNAs in the snRNPs are called small nuclear RNAs or snRNAs. Several snRNPs grouped together form a functional splicing unit called a spliceosome. Spliceosomes are able to recognize short signal sequences in pre-mRNA molecules that identify the boundaries of introns and exons. When a spliceosome has found an intron, it binds correctly, and through formation of a lariat-shaped structure, it cuts the intron out and splices the exons that were on each side of the intron to each other. Genes may have just a few introns, or they may have a dozen or more. Why eukaryotes have introns at all is still an open question, as introns, in general, appear to have no function.
While intron removal and exon splicing are taking place, both ends of maturing mRNAs must also be modified. At the 5′ end (the end with an exposed phosphate) an enzyme adds a modified guanosine nucleotide called 7-methylguanosine. This special nucleotide is added so that ribosomes in the cytoplasm can recognize the correct end of mRNAs, and it probably also prevents the 5′ end of mRNAs from being degraded.
At the 3′ end of maturing mRNAs, another enzyme, called polyadenylase, adds a string of adenine nucleotides. Polyadenylase actually recognizes a special signal in the trailer sequence, at which it cuts and then adds the adenines. The result is what is called a poly-A tail. Initially geneticists did not understand the function of poly-A tails, but now it appears that they protect mRNAs from enzymes in the cytoplasm that could break them down. Essentially, poly-A tails are the main reason mRNAs in eukaryotes survive so much longer than mRNAs in prokaryotes.
Once the modifications have been completed, mRNAs are ready to be exported from the nucleus and will now travel through nuclear pores and enter the cytoplasm, where awaiting ribosomes will translate them, using the RNA code to build polypeptides.
Transcription and Disease
Ordinarily transcription works like a well-oiled machine, and only the right genes are transcribed at the right time so that just the right amount of protein product is produced. Unfortunately, due to the great complexity of the system, problems can occur that lead to disease. It has been estimated that about 15 percent of all genetic diseases may be due to improper intron removal and exon splicing in pre-mRNA molecules. Improper gene expression accounts for many other diseases, including many types of cancer.
Beta-thalassemia, a genetic disorder causing Cooley’s anemia, is caused by a point mutation (a change in a single nucleotide) that changes a cutting and splicing signal. As a result, the mature mRNA has an extra piece of intron, making the mRNA longer and causing a reading frame shift. A reading frame shift causes everything from the mutation forward to be skewed, so that the code no longer codes for the correct amino acids. Additionally, as in the case of Cooley’s anemia, a reading frame shift often introduces a premature stop codon. The gene involved codes for the beta chain of hemoglobin, the protein that carries oxygen in the blood, and this mutation results in a shortened polypeptide that does not function properly.
A single point mutation in a splicing site can have even more far-reaching consequences. In 2000, researchers in Italy discovered an individual who was genetically male (having one X and one Y chromosome) but was phenotypically female. She had no uterus or ovaries and only superficial external female anatomy, making her a pseudohermaphrodite. This condition can be caused by defects either in androgen production or in the androgen receptor. In this case, the defect was a simple point mutation in the androgen receptor gene that led to one intron being retained in the mature mRNA. Within the intron was a stop codon, which meant when the mRNA was translated, a shorter, nonfunctional polypeptide was formed. The subject did show a very small response to androgen, so apparently some of the pre-mRNAs were being cut and spliced correctly, but not enough to produce the normal male phenotype.
The same kinds of mutations as those discussed above can lead to cancer, but mutations that change the level of transcription of proto-oncogenes can also lead to cancer.
Proto-oncogenes
are normal genes involved in regulating the cell cycle, and when these genes are overexpressed they become oncogenes (cancer-causing genes). Overexpression of proto-oncogenes leads to overexpression of other genes, because many proto-oncogenes are transcription factors, signal proteins that interact with molecules controlling intracellular growth and growth factors released by cells to stimulate other cells to divide.
Overexpression can occur when there is a mutation in one of the control regions upstream from a gene. For example, a mutation in the promoter sequence could cause a transcription factor, and thus RNA polymerase, to bind more easily, leading to higher transcription rates. Other control regions, such as enhancer sequences, often far removed from the gene itself, may also affect transcription rates.
Anything that causes the transcription process to go awry will typically have far-reaching consequences. Geneticists are just beginning to understand some of the underlying errors behind a host of genetic diseases, and it should be no surprise that some of them involve how genes are transcribed. Knowing what the problem is, unfortunately, does not usually point to workable solutions. When the primary problem is an excessive rate of transcription, specially designed antisense RNA molecules (RNA molecules that are complementary to mRNA molecules) might be designed that will bind to the overexpressed mRNAs and disable them. This approach is still being tested. In the case of point mutations that derail the cutting and splicing process, the only solution may be gene therapy, a technique still not considered technically possible and not expected to be feasible for some time to come.
Key terms
messenger RNA (mRNA)
:
the form of RNA that contains the coding instructions used to make a polypeptide by ribosomes
RNA polymerase
:
the enzyme that transcribes RNA using a strand of DNA as a template
transcription
:
the process that converts DNA code into a complementary strand of RNA (mRNA) containing code that can be interpreted by ribosomes
translation
:
the process, mediated by ribosomes, in which the genetic code in an mRNA is used to produce a polypeptide, the ultimate product of structural genes
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