RPB4, A SUBUNIT OF RNA-POLYMERASE-II, ENABLES THE ENZYME TO TRANSCRIBE AT TEMPERATURE EXTREMES IN-VITRO

Rpb4 is a subunit of Saccharomyces cerevisiae RNA polymerase II (Pol I I). It associates with the polymerase preferentially in stationary pha se and is essential for some stress responses. Using the promoter-inde pendent initiation and chain elongation assay, we monitored Pol II enz ymatic activity in cell extracts. We show here that Rpb4 is required f or the polymerase activity at temperature extremes (10 and 35 degrees C). In contrast, at moderate temperature (23 degrees C) Pol II activit y is independent of Rpb4. These results are consistent with the role p reviously attributed to Rpb4 as a subunit whose association with Pol I I helps Pol II to transcribe during extreme temperatures. The enzymati c inactivation of Pol II lacking Rpb4 at the nonoptimal temperature wa s prevented by the addition of recombinant Rpb4 produced in Escherichi a coli prior to the in vitro reaction assay. This finding suggests tha t modification of Rpb4 is not required for its functional association with the other Pol II subunits. Sucrose gradient and immunoprecipitati on experiments demonstrated that Rpb4 is present in the cell in excess over the Pol II complex during all growth phases. Nevertheless, the r escue of Pol II activity at the nonoptimal temperature by Rpb4 is poss ible only when cell extracts are obtained from postlogarithmic cells, not from logarithmically growing cells. This result suggests that Pol II molecules should be modified in order to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes predominant in stationary phase.