Formation and stability of the 5 S RNA transcription complex

Abstract

5 S ribosomal RNA in Xenopus has been shown to be transcribed in vitro from 5 S RNA genes that remain stably associated with required transcription factors through multiple rounds of transcription (Bogenhagen, D. F., Wormington, W. M., and Brown, D. D. (1982) Cell 28, 413-421). We have studied the formation and stability of these "transcription complexes" by using cloned 5 S RNA genes immobilized on cellulose as templates for the assembly of complexes in crude extracts. RNA polymerase III is the least tightly bound component required for transcription of 5 S RNA genes. All other factors remain bound in 1 M NaCl, even though transcription complexes do not form at salt concentrations as low as 0.25 M. RNA polymerase III dissociates from transcription complexes as a result of RNA synthesis and is capable of reassociating with complexes to support additional rounds of transcription. A 5 S-specific positive transcription factor (factor A) and two crude phosphocellulose column fractions (B and C) are also required for 5 S RNA synthesis in vitro (Engelke, D. R., Ng, S.-Y., Shastry, B. S., and Roeder, R. G. (1980) Cell 19, 717-728; Segall, J., Matsui, T., and Roeder, R. G. (1980) J. Biol. Chem. 255, 11986-11991; Shastry, B. S., Ng, S.-Y., and Roeder, R. G. (1982) J. Biol. Chem. 257, 12979-12986). Fraction B stably interacts with 5 S RNA genes to form a stable, active complex only after the template has first been incubated with factor A and fraction C. In contrast, either factor A or fraction C can stably associate with 5 S RNA genes in the absence of other factors. The activities of fractions B and C are removed from solution as a result of transcription complex formation, suggesting the factors in these fractions act stoichiometrically. The rate-limiting step in complex formation is carried out by fraction B, which accounts for the lag in transcription activity observed in crude extracts.