Transcriptional activation of a gene involves an orchestrated recruitment of components of the basal transcription machinery and intermediate factors, concomitant with an alteration in local chromatin structure generated by posttranslational modifications of histone tails and nucleosome remodeling. We provide here a comprehensive picture of events resulting in transcriptional activation of a gene, through evaluating the estrogen receptor-alpha (NR3A1) target pS2 gene promoter in MCF-7 cells. This description integrates chromatin remodeling with a kinetic evaluation of cyclical networks of association of 46 transcription factors with the promoter, as determined by chromatin immunoprecipitation assays. We define the concept of a "transcriptional clock" that directs and achieves the sequential and combinatorial assembly of a transcriptionally productive complex on a promoter. Furthermore, the unanticipated findings of key roles for histone deacetylases and nucleosome-remodeling complexes in limiting transcription implies that transcriptional activation is a cyclical process that requires both activating and repressive epigenetic processes.

A new isoform of the human estrogen receptor-alpha (hER-a) has been identi®ed and characterized. This 46 kDa isoform (hERa46) lacks the N-terminal 173 amino acids present in the previously characterized 66 kDa isoform (hERa66). hERa46 is encoded by a new class of hER-a transcript that lacks the ®rst coding exon (exon 1A) of the ER-a gene. We demonstrated that these D1A hER-a transcripts originate from the E and F hER-a promoters and are produced by the splicing of exon 1E directly to exon 2. Functional analysis of hERa46 showed that, in a cell context sensitive to the transactivation function AF-2, this receptor is an effective ligand-inducible transcription factor. In contrast, hERa46 is a powerful inhibitor of hERa66 in a cell context where the transactivating function of AF-1 predominates over AF-2. The mechanisms by which the AF-1 dominantnegative action is exerted may involve heterodimerization of the two receptor isoforms and/or direct competition for the ER-a DNA-binding site. hERa66/ hERa46 ratios change with the cell growth status of the breast carcinoma cell line MCF7, suggesting a role of hERa46 in cellular proliferation.

Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

SummaryTo better understand yeast ribosome synthesis, we developed techniques for the rapid harvesting and analysis of metabolically labeled cultures. Modeling of the resulting kinetic data allowed predicted lifetimes and processing patterns to be compared with the experimental data. This supported a transcription time for the 35S primary transcripts of ∼170 s at 30°C (∼40 nt s−1), with a high fraction (∼70%) of nascent transcripts cleaved at the early processing sites that generate the 20S precursor to the 18S rRNA. This level of nascent transcript cleavage apparently conflicted with previous reports that modification of yeast pre-rRNA exclusively occurred on released transcripts. A second round of high-resolution kinetic labeling showed that 20S pre-rRNA predominately undergoes methylation as nascent transcripts, whereas the 27S precursor to the 25S/5.8S rRNAs was partially methylated on the nascent transcript. The results demonstrate that quantitative analyses of pre-rRNA processing can yield important biological insights.