The carboxyl end of RPB1, the largest subunit of RNA polymerase II, contains a stretch of seven amino acids that is nearly precisely repeated multiple times. Neither RNA polymerase I nor III contains these repeating units. This heptapeptide repeat, with a consensus sequence of Tyr Ser-Pro-Thr-Ser-Pro-Ser, is known as the carboxy-terminal domain (CTD). Yeast RNA polymerase II contains 26 or more repeats, vertebrate enzymes have 52 repeats, and an intermediate number of repeats occur in RNA polymerase II from nearly all other eukaryotes. The CTD is critical for viability, and at least 10 copies of the repeat must be present for yeast to survive.

In vitro experiments with model promoters first showed that RNA polymerase II molecules that initiate transcription have a nonphosphorylated CTD. Once the polymerase initiates transcription and begins to move away from the promoter, many of the serine and some tyrosine residues in the CTD are phosphorylated. Analysis of polytene chromosomes from Drosophila salivary glands prepared just before molting of the larva, a time of active transcription, indicates that the CTD is also phosphorylated during in vivo transcription. The large chromosomal “puffs” induced at this time in development are regions where the genome is very actively transcribed. Staining with antibodies specific for the phosphorylated or nonphosphorylated CTD demonstrated that RNA polymerase II associated with the highly transcribed puffed regions contains a phosphorylated CTD (Figure 1).

EXPERIMENTAL FIGURE 1 Antibody staining demonstrates that the carboxy-terminal domain of RNA polymerase II is phosphorylated during in vivo transcription. Salivary-gland polytene chromosomes were prepared from Drosophila larvae just before they molted. The preparation was treated with a rabbit antibody specific for phosphorylated CTD and with a goat antibody specific for nonphosphorylated CTD. The prepa ration was then stained with fluorescein-labeled anti-goat antibody (green) and rhodamine-labeled anti-rabbit antibody (red). Thus polymerase molecules with a nonphosphorylated CTD stained green, and those with a phosphorylated CTD stained red. The molting hormone ecdysone induces very high rates of transcription in the puffed regions labeled 74EF and 75B; note that only phosphorylated CTD is present in these regions. Smaller puffed regions transcribed at high rates are also visible. Nonpuffed sites that stained red (up arrow) or green (horizontal arrow) are also indicated, as is a site staining both red and green, producing a yellow color (down arrow). [From J. R. Weeks et al., “Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing,” Genes & Development, 1993, 7(12A):2329–44; courtesy of J. R. Weeks and A. L. Greenleaf; republished with permission from Cold Spring Harbor Press.]

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مواضيع ذات صلة

Three Eukaryotic RNA Polymerases Catalyze Formation of Different RNAs

Regulatory Elements in Eukaryotic DNA Are Found Both Close to and Many Kilobases Away from Transcription Start Sites

Control of Gene Expression in Bacteria

Nucleic Acid Electrophoresis

Analysing Genes and Gene Expression

Screening Gene Libraries

Hybridisation and Gene Probes

Cloning Vectors: Vectors Used in Eukaryotes

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes

Centromere Sequences Vary Greatly in Length and Complexity

Interphase Polytene Chromosomes Arise by DNA Amplification

Cloning Vectors : Cosmid-Based Vectors