A monoclonal antibody directed against a small subunit of RNA polymerase B blocks the initiation step

Abstract

Monoclonal antibodies directed against RNA polymerase B of the fungus Podospora were probed for their inhibitory effect on enzyme activity in vitro. From nine antibodies directed against various subunits of the enzyme only two of them totally inhibit RNA polymerase B activity in a non-selective transcription system. Enzymes A and C are not inhibited at all. The two antibodies recognize the same small subunit (B11) of Podosporu enzyme B. One of these antibodies has been used as a specific inhibitor to study the function of the B11 subunit. Enzyme activity is partially protected from the inhibition when the enzyme is previously incubated with the template. The antibody does not interfere with enzyme-DNA complex formation but blocks the initiation process in a transcription system using a dinucleotide as initiator. Elongation of RNA chains does not appear to be affected. Also the antibody cross-reacts with yeast and calf thymus RNA polymerases B and has some, but less, effect on initiation by these enzymes. It is suggested the antigenic site corresponding to this antibody may overlap the catalytic initiation site on the native form of RNA polymerase B.In eukaryotes the three classes of genes encoding rRNA, heterogenous nuclear RNA, and 5s RNA and tRNA are transcribed by three distinct RNA polymerases termed A (or I), B (or 11) and C (or 111) [l, 21. They have so complex a subunit structure that very little is known about the part played by the individual subunits in enzyme function.Few specific RNA polymerase inhibitors are available and genetic studies failed to give any information. Some of the subunits (possibly the common subunits of the three enzymes) may be responsible for the basic steps of the transcription reaction while the others may be implicated in the specificity of the transcription or in the regulation of transcriptional activity during differentiation processes. Specific probes for each subunit will greatly help in investigating these complex enzymes.Antibodies directed against the different subunits of yeast RNA polymerases [3] have allowed the precise structural characterization of yeast transcriptional apparatus. Functional studies have shown that most of the antibodies have an inhibitory effect in a non-specific transcription system, arguing in favour of the idea that all the subunits take part in the work of the enzyme. However, these studies have been carried out with polyspecific antibodies. For such functional studies more specific probes may be obtained with the use of monoclonal antibodies. calf thymus [8, 91. Few of these antibodies exhibit inhibitory effects in a non-specifi; transcription system. This does not exclude the possibility that they may inhibit specific transcription in appropriate systems as shown for a monoclonal antibody directed against RNA polymerase B from calf thymus [lo].We previously reported the characterization of nine monoclonal antibodies directed against RNA polymerase B from the fungus Podospora [ll]. Here we report the use of one of these antibodies as a spec...

Osteoarthritis‐related changes in human articular cartilage

“…The results in Figure 5 show that the binding to hyaluronic acid was decreased when the proteoglycan monomers were preincubated with 5 pg and 50 pg of MAb 8-E-10. No change in binding was observed after preincubation with a nonspecific anti-RNA polymerase B antibody (IgGT) (24). This result was confirmed using proteoglycan subunits from osteoarthritic cartilage (results not shown).…”

“…Polypeptide bands were excised from the gels after staining with Coomassie blue or toluidine blue. Proteins were eluted from gel pieces by incubating them in 50 pl of 100 mM Tris HCI, pH 8, 0.1% (w/v) sodium dodecyl sulfate (SDS) for 48 hours, as previously described (24). Supernatants were collected by centrifugation of samples for 2 minutes at 10,000g.…”

RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth.

“…It is not clear whether all of these polypeptides are functional subunits, since some of the smaller polypeptides may be degradation products or proteins that simply copurify with RNA polymerase II. However, the conservation of the small proteins among eucaryotic RNA polymerase II enzymes (20) and the fact that antibodies directed against many of the small RNA polymerase II subunits can inhibit transcription in vitro (4,25,26) suggest that these proteins do have a role in the mRNA transcription apparatus.…”