Visual Analysis of the Yeast 5S rRNA Gene Transcriptome: Regulation and Role of La Protein

Abstract

5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, ∼132 nucleotides (nt), were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a fourfold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (presence of rapamycin or postexponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. The polymerase III elongation rate was estimated to be in the range of 60 to 75 nt/s, with a reinitiation interval of ∼1.2 s. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on the amount or size of 5S RNA produced yet resulted in more polymerases per gene on average, consistent with a non-rate-limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

The work was supported by NIH Public Health Service grants GM-63952 (to A.L.B.), GM-35949 (to M.N.), and GM-48410 (to S.L.W.) and a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research (to D.A.S.).

We thank A. van Hoof and R. Parker for yeast strains.