Eukaryotic transcription uses three distinct RNA polymerases, which are specialized for different RNAs. RNA polymerase I makes Ribosomal RNAs, RNA polymerase II makes messenger RNAs, and RNA polymerase III makes small, stable RNAs such as transfer RNAs and 5S ribosomal RNA. Eukaryotic RNA polymerases are differentiated by their sensitivity to the toxic compound, α ‐amanitin, the active compound in the poisonous mushroom Aminita phalloides, or “destroying angel.” RNA polymerase I is not inhibited by α‐amanitin, RNA polymerase II is inhibited at very low concentrations of the drug, and RNA polymerase III is inhibited at high drug concentrations.
Eukaryotic transcription is dependent on several sequence and structural features. First, actively transcribing genes have a “looser,” more accessible chromatin structure. The nucleosomes are not as condensed as in other forms of chromatin, especially heterochromatin, and they often do not contain histone H1. The DNA in the promoter region at the 5′ end of the gene may not be bound into nucleosomes at all. In this way, the promoter sequences are available for binding to protein transcription factors—proteins that bind to DNA and either repress or stimulate transcription. In addition to promoter sequences, other nucleotide sequences termed enhancers can affect transcription efficiency. Enhancers bind to specialized protein factors and then stimulate transcription. The difference between enhancers and factor‐binding promoters depends on their site of action. Unlike promoters, which only affect sequences immediately adjacent to them, enhancers function even when they are located far away (as much as 1,000 base pairs away) from the promoter. Both enhancer‐binding and promoter‐binding transcription factors recognize their appropriate DNA sequences and then bind to other proteins—for example, RNA polymerase, to help initiate transcription. Because enhancers are located so far from the promoters where RNA polymerase binds, enhancer interactions involve bending the DNA to make a loop so the proteins can interact.
Most of the RNA made in the cell is ribosomal RNA. The large and small subunit RNAs are synthesized by RNA polymerase I. Ribosomal RNA is made in a specialized organelle, the nucleolus, which contains many copies of the rRNA genes, a correspondingly large number of RNA polymerase I molecules, and the cellular machinery that processes the primary transcripts into mature rRNAs. RNA polymerase I is the most abundant RNA polymerase in the cell, and it synthesizes RNA at the fastest rate of any of the polymerases. The genes for rRNA are present in many copies, arranged in tandem, one after the other. Each transcript contains a copy of each of three rRNAs: the 28S and 5.8S large subunit RNAs and the small subunit 18S RNA, in that order. The rRNA promoter sequences extend much further upstream than do prokaryotic promoters. The transcription of rRNA is very efficient. This is necessary because each rRNA transcript can only make one ribosome, in contrast to the large number of proteins that can be made from a single mRNA.
The individual ribosomal RNAs must be processed from the large precursor RNA that is the product of transcription. The primary transcript contains small and large subunit RNAs in the order: 28S—5.8S—18S. Processing involves the modification of specific nucleotides in the rRNA, followed by cleavage of the transcript into the individual RNA components. See Figure 1 .
RNA polymerase II transcribes messenger RNA and a few other small cellular RNAs. Class II promoters are usually defined by their sensitivity to α‐amanitin. Like prokaryotic promoters, many class II promoters contain two conserved sequences, called the CAAT and TATA boxes. The TATA box is bound by a specialized transcription factor called TBP (for TATA‐Binding‐Factor). Binding of TBP is required for transcription, but other proteins are required to bind to the upstream (and potentially downstream) sequences that are specific to each gene. Like prokaryotic transcripts, eukaryotic RNAs are initiated with a nucleoside triphosphate. Termination of eukaryotic mRNA transcription is less well understood than is termination of prokaryotic transcription, because the 3′ ends of eukaryotic mRNAs are derived by processing. See Figure 2.
RNA polymerase III transcribes 5S rRNA and tRNA genes. The “promoter” of these transcripts can actually be located inside the gene itself, in contrast to all the other promoters discussed earlier. See Figure .
The 5′ sequence is not essential for accurate transcription initiation. When the region extending from the 5′ end of the gene (that is, the part that would normally be considered to be the promoter) is deleted, RNA synthesis is carried out just as efficiently as on the native gene. The new 5′ end of the transcript is complementary to whatever sequences take the place of the natural ones. Furthermore, initiation is only affected when sequences within the 5S rRNA gene are disrupted. The molecular explanation for this phenomenon is as follows:
- A protein factor binds to the 5S rRNA gene. Binding is at the internal sequence that is required for accurate initiation.
- The bound factor then interacts with RNA polymerase III, which is then capable of initiation. During transcription, the multiple protein factors (called TFIIIs) remain bound to the transcribing gene.