The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.Nuclear transcription is repressed during mitosis (4,7,9,31,45,46). In mammalian cells, all RNA synthesis stops by midprophase before any disintegration of the nuclear membrane is apparent, and it does not resume again until late in telophase (31). This repression may be necessary to allow chromosomal condensation and division to occur without interference from the transcriptional apparatus. However, very little is known about the molecular mechanisms responsible for transcriptional inhibition at mitosis. An important step toward characterizing this process came with the recent demonstration that mitotic repression can be mimicked in vitro by using Xenopus egg extracts (15). Hartl et al. (15) showed that extracts shifted to a mitotic state by the addition of recombinant cyclin B exhibit a marked decrease in ability to transcribe tRNA or 5S RNA genes relative to untreated interphase extracts. This happens even if active transcription complexes are preassembled on the promoters of these genes (15). The in vitro inhibition does not require nucleosome deposition on the template and occurs in the presence of the topoisomerase II inhibitor VM-26, which prevents the complete assembly of chromosomes into metaphase structures. It appears to involve phosphorylation-dependent inactivation of one or more components of the transcription machinery that are required for tRNA and 5S RNA synthesis (15).tRNA and 5S RNA genes are transcribed by RNA polymerase III (Pol III). The proteins involved in this process have been studied extensively (reviewed in references 51 and 59). Recruitment of Pol III to specific promoter sites requires a factor called TFIIIB, which is a multisubunit complex containing the TATA-binding protein (TBP) and associated polypeptides (reviewed in references 16 and 34). Since most Pol III promoters contain no TATA sequence and cannot be recognized directly by TBP (19,22,53), TFIIIB is normally recruited via protein-protein interactions with an assembly factor called TFIIIC that binds downstream of the initiation site (reviewed in references 16, 51, and 59).We have investigated how the human Pol III transcription apparatus behaves at mitosis by comparing extracts made from asynchronous HeLa cells with those of cells synchronized in M phase of the active gro...