In exponentially growing Saccharomyces cerevisiae cells, rRNA synthesis is determined by the summed RNA polymerase I loading rate rather than by the number of active genes

Abstract

Genes encoding rRNA are multicopy and thus could be regulated by changing the number of active genes or by changing the transcription rate per gene. We tested the hypothesis that the number of open genes is limiting rRNA synthesis by using an electron microscopy method that allows direct counting of the number of active genes per nucleolus and the number of polymerases per active gene. Two strains of Saccharomyces cerevisiae were analyzed during exponential growth: a control strain with a typical number of rRNA genes ( approximately 143 in this case) and a strain in which the rRNA gene number was reduced to approximately 42 but which grows as well as controls. In control strains, somewhat more than half of the genes were active and the mean number of polymerases/gene was approximately 50 +/- 20. In the 42-copy strain, all rRNA genes were active with a mean number of 100 +/- 29 polymerases/gene. Thus, an equivalent number of polymerases was active per nucleolus in the two strains, though the number of active genes varied by twofold, showing that overall initiation rate, and not the number of active genes, determines rRNA transcription rate during exponential growth in yeast. Results also allow an estimate of elongation rate of approximately 60 nucleotides/s for yeast Pol I and a reinitiation rate of less than 1 s on the most heavily transcribed genes.

FIG. 1.

Comparison of the reduced gene copy strain, NOY886, with a control strain, NOY1051, for size of chromosome XII (A), rDNA copy number (B), and rRNA synthesis rate (C). (A) Chromosomal DNA was isolated and analyzed by CHEF electrophoresis. An ethidium bromide-stained gel is shown. Positions of chromosome XII are indicated by a white dot. Size markers (lane M) are Hansenula wingei chromosomes. When these markers were used, the sizes of chromosome XII estimated for NOY1051^143C and NOY886^42C were approximately 2.4 and 1.48 Mb, respectively, which correspond to rDNA repeat numbers of 143 and 42, respectively. (B) For Southern analysis, DNA was isolated and digested with HindIII and PstI and digests were subjected to agarose gel electrophoresis, followed by transfer to a nylon membrane, and were then subjected to hybridization first with a LEU2 probe (a 598-bp ClaI-EcoRV fragment) and then with an rDNA probe (a 613-bp SmaI-EcoRV fragment). An autoradiogram is shown. Radioactivity in each band was quantified with a PhosphorImager. The ratios of the values for rDNA after normalization to the values for reference LEU2 DNA (the LEU2 gene used for disruption of RPA135) were calculated. The ratios of the amount of rDNA for NOY1051^143C to that for NOY886^42C were then calculated, and the average ratio from five independent experiments was 3.0 ± 0.3. (C) RNA was isolated from cells, and 2.5 and 5.0 μg were used to determine the amounts of the 5′ end of 35S rRNA by primer extension. Radioactive bands visualized by a PhosphorImager are shown. The ratios of the radioactivity in the band for NOY886^42C to that for NOY1051^143C were calculated. The average ratio from three independent experiments was 1.1 ± 0.1.

FIG. 2.

The yeast rRNA gene repeat unit. (A) Schematic of four tandem rRNA gene repeats showing transcriptionally active and inactive units. The endogenous S. cerevisiae RDN1 locus on chromosome XII contains ∼150 of these repeats. (B) Schematic of one gene spacer unit, including the Pol I-transcribed 35S rRNA gene (long thick line), the Pol III-transcribed 5S rRNA gene (short thick line), and nontranscribed spacers (thin lines). (C) A representative electron micrograph of the rRNA gene spacer unit from the control JS772 strain is shown, aligned with the gene map in panel B. Long arrow indicates direction of transcription for the Pol I gene. It was identified as a Pol I gene by its tandem repetition (not shown), its expected length (corresponding to ∼6.7 kb), and the characteristic terminal knobs at the 5′ ends of the nascent rRNA transcripts. The short arrow indicates the structure frequently seen at the position of the 5S gene. Bar = 0.4 μm.

FIG. 3.

More genes are active per nucleolus in the control strain, NOY1051^143C, than in the 40-copy strain, NOY886^42C, as determined by direct gene counts. (A to D) Four micrographs, at the same magnification, showing dispersed nucleolar contents from NOY886^42C (A and B) and NOY1051^143C (C and D) and from cells known or interpreted to be in G₁ (A and C) or G₂ (B and D). The chromatin in B has been released from a budded cell (seen at right in panel B), indicating that it is in the G₂ stage of the cell cycle. The rectangular boxed areas in panels A to D are shown at higher magnification in panels A′ to D′, better displaying examples of the characteristic active rRNA genes that are present in each nucleolar cluster. In panels A and B, some nonnucleolar chromatin is present (thin arrows) as well as nucleolar chromatin (thick arrows), the latter of which appears as a darker grey area in micrographs at this low magnification. (The dark fat rods in panels A and B are contaminating bacteria). A-trace and B-trace are schematic tracings of the DNA template of the active genes in the nucleoli in panels A and B. In panels C and D, essentially all of the dispersed chromatin shown is nucleolar. Note the increasing size of the nucleolar gene cluster as one goes from panels A to D. Bar in panel D = 2 μm; bar in panel D′ = 1 μm; panels A to D are at same magnification. (E) The active rRNA gene number per nucleolus was estimated for NOY1051^143C (n = 28) and for NOY886^42C (n = 44) by using micrographs shown in panels A to D and additional micrographs, and the results are shown graphically.

FIG. 4.

A higher percentage of rRNA genes is active in NOY886^42C as determined by analysis of activity of contiguous gene repeats. Examples of genes repeated in tandem from NOY886^42C (A and B) and NOY1051^143C (C to E). The black arrows indicate active genes and direction of transcription. The grey arrows indicate inactive genes, which were identified as a nontranscribed region immediately upstream or downstream of an active gene. Inactive genes were very rarely seen in NOY886^42C (one example is shown by grey arrow in panel B) but were common in NOY1051^143C (grey arrows in panels C to E). More than 99% of rRNA genes were active in NOY886^42C by this analysis, while 67% of genes were active in NOY1051^143C (see text). The micrograph in panel A displays a complete NOY886^42C RDN1 locus, containing the tandem rRNA genes from one chromosome XII, with both ends of the locus identifiable (arrowheads), and with ∼42 active genes. The inset in panel A shows two of these genes at a higher magnification; the asterisk indicates a transcript-free gap in the middle of one gene. In both panels D and E, one gene is marked with a black arrow and pound sign. These are active genes with low polymerase density (discussed in text). Bar in panel A = 2 μm; bar in panel C = 1 μm; panels B to E are at approximately the same magnification.

FIG. 5.

The number of polymerases per gene is higher in the strain with fewer genes. (A) The average number of polymerases per gene is lower in the two control strains that have a typical number of genes (NOY1051^143C and JS772) than in the ∼42-copy strain NOY886^42C. In all strains, there is a wide range in the number of polymerases per gene. (B and C) Representative rRNA genes from NOY886^42C (B) and NOY1051^143C (C) showing genes with an average number of polymerases. Bar = 0.5 μm. (D and E) Representative clusters of dispersed genes from NOY886^42C (D) and NOY1051^143C (E) showing the more prominent polymerase backbones on the genes in the ∼42-copy strain, due to the very dense packing of polymerases on the genes. Bar = 0.5 μm.