A novel 15-subunit complex with the capacity to remodel the structure of chromatin, termed RSC, has been isolated from S. cerevisiae on the basis of homology to the SWI/SNF complex. At least three RSC subunits are related to SWI/SNF polypeptides: Sth1p, Rsc6p, and Rsc8p are significantly similar to Swi2/Snf2p, Swp73p, and Swi3p, respectively, and were identified by mass spectrometric and sequence analysis of peptide fragments. Like SWI/SNF, RSC exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structure. RSC is, however, at least 10-fold more abundant than SWI/SNF complex and is essential for mitotic growth. Contrary to a report for SWII/SNF complex, no association of RSC (nor of SWI/SNF complex) with RNA polymerase II holoenzyme was detected.

RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle to naked DNA. The newly formed octamer-DNA complex is identical with a nucleosome in all respects. The reaction requires ATP and involves an activated RSC-nucleosome intermediate. The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule.

RSC, an abundant, essential chromatin-remodeling complex, related to SWI/SNF complex, binds nucleosomes and naked DNA with comparable affinities, as shown by gel shift analysis. The RSC-nucleosome complex is converted in the presence of ATP to a slower migrating form. This activated complex exhibits greatly increased susceptibility to endo- and exonucleases but retains a full complement of histones. Activation persists in the absence of ATP, and on removal of RSC, the nucleosome is released in an altered form, with a diminished electrophoretic mobility, greater sedimentation rate, and marked instability at elevated ionic strength. The reaction is reversible in the presence of RSC and ATP, with conversion of the altered form back to the nucleosome.

(5) by binding to domain 1 (6) to activate domain 6 (7). Previous work (S. C. Boulanger, et aL, unpublished data) has identified sites that are important for D5 function, but that study could not provide a specific diagnosis for the functional defects of inactive variants. We have applied a trans assay by using a 36-nt D5 RNA that activates group II intron transcripts deleted for D5 (AD5; see Fig. 1) (7,9). Accurate hydrolysis at the 5' splice junction is the most efficient reaction, although branching and the second step of splicing also occur. The Michealis constant (Km) measures the affinity of D5 binding to the AD5 substrate, while the turnover number for 5' splice junction hydrolysis (kcat) gauges D5 catalytic action (see Fig. 2 and refs. 9 and 10). We describe here the consequences of single-base substitutions affecting the three most highly conserved base pairs of a crucial segment (A2-G3-C4 paired with G31-U32-U33) in D5 from a group II self-splicing intron. We also surveyed double substitutions, including several Watson-Crick base pair replacements, affecting these three conserved base pairs. MATERIALS AND METHODSD5 and AD5 Transcripts. Wild-type and variant D5s had a 5'-GG-3' leader from the T7 RNA polymerase promoter of the synthetic DNA templates (9, 11). Radioactive AD5 substrate was transcribed from plasmid pJD20A5 (7). Transcripts were purified by denaturing polyacrylamide gel electrophoresis, and concentrations were estimated by spectrophotometry in water by using extinction coefficients at 260 nm of 7500 M-'cm-l for D5 and 10,000 M-'-cm-1 for AD5.

RSC and SWI/SNF chromatin-remodeling complexes were previously reported to generate a stably altered nucleosome. We now describe the formation of hybrids between nucleosomes of different sizes, showing that the stably altered structure is a noncovalent dimer. A basis for dimer formation is suggested by an effect of RSC on the supercoiling of closed, circular arrays of nucleosomes. The effect may be explained by the interaction of RSC with DNA at the ends of the nucleosome, which could lead to the release 60--80 bp or more from the ends. DNA released in this way may be trapped in the stable dimer or lead to alternative fates such as histone octamer transfer to another DNA or sliding along the same DNA molecule.