Eukaryotic RNA polymerases: Characterization and function
▶Eukaryotic RNA polymerases: The mechanism of eukaryotic transcription is similar to that in prokaryotes. However, the large number of polypeptides associated with the eukaryotic transcription machinery makes it far more complex. Three different RNA polymerase complexes are responsible for the transcription of different types of eukaryotic genes. The different RNA polymerases were identified by chromatographic purification of the enzymes and elution at different salt concentrations. Each RNA polymerase has a different sensitivity to the fungal toxin α -amanitin and this can be used to distinguish their activities.
- RNA polymerase I (RNA Pol I) transcribes most rRNA genes. It is located in the nucleoli and is insensitive to α-amanitin.
- RNA polymerase II (RNA Pol II) transcribes all protein-coding genes and some small nuclear RNA (snRNA) genes. It is located in the nucleoplasm and is very sensitive to α-amanitin.
- RNA polymerase III (RNA Pol III) transcribes the genes for tRNA, 5S rRNA, U6 snRNA and certain other small RNAs. It is located in the nucleoplasm and is moderately sensitive to α -amanitin. In addition to these nuclear enzymes, eukaryotic cells contain additional polymerases in mitochondria and chloroplasts.
▶RNA polymerase subunits: All three polymerases are large enzymes containing 12 or more subunits. The genes encoding the two largest subunits of each RNA polymerase have homology (related DNA coding sequences) to each other. All of the three eukaryotic polymerases contain subunits which have homology to subunits within the E. coli core RNA polymerase α2 β β’. The largest subunit of each eukaryotic RNA polymerase is similar to the β’ subunit of the E. coli polymerase, and the second largest subunit is similar to the β subunit which contains the active site of the E. coli enzyme. The functional significance of this homology is supported by the observation that the second largest subunits of the eukaryotic RNA polymerases also contain the active sites. Two subunits which are common to RNA Pol I and RNA Pol III, and a further subunit which is specific to RNA Pol II, have homology to the E. coli RNA polymerase α subunit. At least five other smaller subunits are common to the three different polymerases. Each polymerase also contains an additional four to seven subunits which are only present in one type.
▶Eukaryotic RNA polymerase activities: Like bacterial RNA polymerases, each of the eukaryotic enzymes catalyzes transcription in a 5’ to 3’ direction and synthesizes RNA complementary to the antisense template strand. The reaction requires the precursor nucleotides ATP, GTP, CTP and UTP and does not require a primer for transcription initiation. The purified eukaryotic RNA polymerases, unlike the purified bacterial enzymes, require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription.
▶The CTD of RNA Pol II : The carboxyl end of RNA Pol II contains a stretch of seven amino acids that is repeated 52 times in the mouse enzyme and 26 times in yeast. This heptapeptide has the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser and is known as the carboxyterminal domain or CTD. These repeats are essential for viability. The CTD sequence may be phosphorylated at the serines and some tyrosines. In vitro studies have shown that the CTD is unphosphorylated at transcription initiation, but phosphorylation occurs during transcription elongation as the RNA polymerase leaves the promoter. Since RNA Pol II catalyzes the synthesis of all of the eukaryotic protein-coding genes, it is the most important RNA polymerase for the study of differential gene expression. The CTD has been shown to be an important target for differential activation of transcription elongation and enhances capping and splicing.
▶Eukaryotic RNA polymerases: Three eukaryotic polymerases transcribe different sets of genes. Their activities are distinguished by their different sensitivities to the fungal toxin α-amanitin.
- RNA polymerase I is located in the nucleoli. It is responsible for the synthesis of the precursors of most rRNAs.
- RNA polymerase II is located in the nucleoplasm and is responsible for the synthesis of mRNA precursors and some small nuclear RNAs.
- RNA polymerase III is located in the nucleoplasm. It is responsible for the synthesis of the precursors of 5S rRNA, tRNAs and other small nuclear and cytosolic RNAs.
▶RNA polymerase subunits: Each RNA polymerase has 12 or more different subunits. The largest two subunits are similar to each other and to the β’ and β subunits of coli RNA polymerase. Other subunits in each enzyme have homology to the α subunitof the E. coli enzyme. Five additional subunits are common to all three polymerases, and others are polymerase specific.
▶Eukaryotic RNA polymerase activities: Like prokaryotic RNA polymerases, the eukaryotic enzymes do not require a primer and synthesize RNA in a 5’ to 3’ Unlike bacterial polymerases, they require accessory factors for DNA binding.
▶The CTD of RNA Pol II: The largest subunit of RNA polymerase II has a seven amino acid repeat at the C terminus called the carboxy-terminal domain (CTD). This sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser, is repeated 52 times in the mouse RNA polymerase II and is subject to phosphorylation.