Journey of a molecular biologist.

My journey into a research career began in fermentation biochemistry in an applied science department during the difficult post-World War II time in Japan. Subsequently, my desire to do research in basic science developed. I was fortunate to be a postdoctoral fellow in the United States during the early days of molecular biology. From 1957 to 1960, I worked with three pioneers of molecular biology, Sol Spiegelman, James Watson, and Seymour Benzer. These experiences helped me develop into a basic research scientist. My initial research projects at Osaka University, and subsequently at the University of Wisconsin, Madison, were on the mode of action of colicins as well as on mRNA and ribosomes. Following success in the reconstitution of ribosomal subunits, my efforts focused more on ribosomes, initially on the aspects of structure, function, and in vitro assembly, such as the construction of the 30S subunit assembly map. After this, my laboratory studied the regulation of the synthesis of ribosomes and ribosomal components in Escherichia coli. Our achievements included the discovery of translational feedback regulation of ribosomal protein synthesis and the identification of several repressor ribosomal proteins used in this regulation. In 1984, I moved to the University of California, Irvine, and initiated research on rRNA transcription by RNA polymerase I in the yeast Saccharomyces cerevisiae. The use of yeast genetics combined with biochemistry allowed us to identify genes uniquely involved in rRNA synthesis and to elucidate the mechanism of initiation of transcription. This essay is a reflection on my life as a research scientist.

The transcription elongation factor Spt5 influences transcription by RNA polymerase I positively and negatively.

Spt5p is a universally conserved transcription factor that plays multiple roles in eukaryotic transcription elongation. Spt5p forms a heterodimer with Spt4p and collaborates with other transcription factors to pause or promote RNA polymerase II transcription elongation. We have shown previously that Spt4p and Spt5p also influence synthesis of ribosomal RNA by RNA polymerase (Pol) I; however, previous studies only characterized defects in Pol I transcription induced by deletion of SPT4. Here we describe two new, partially active mutations in SPT5 and use these mutant strains to characterize the effect of Spt5p on Pol I transcription. Genetic interactions between spt5 and rpa49Δ mutations together with measurements of ribosomal RNA synthesis rates, rDNA copy number, and Pol I occupancy of the rDNA demonstrate that Spt5p plays both positive and negative roles in transcription by Pol I. Electron microscopic analysis of mutant and WT strains confirms these observations and supports the model that Spt4/5 may contribute to pausing of RNA polymerase I early during transcription elongation but promotes transcription elongation downstream of the pause(s). These findings bolster the model that Spt5p and related homologues serve diverse critical roles in the control of transcription.

Transcription of multiple yeast ribosomal DNA genes requires targeting of UAF to the promoter by Uaf30.

Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by approximately 70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.

Visual analysis of the yeast 5S rRNA gene transcriptome: regulation and role of La protein.

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, approximately 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 approximately 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.

Transcription elongation by RNA polymerase I is linked to efficient rRNA processing and ribosome assembly.

The synthesis of ribosomes in eukaryotic cells is a complex process involving many nonribosomal protein factors and snoRNAs. In general, the processes of rRNA transcription and ribosome assembly are treated as temporally or spatially distinct. Here, we describe the identification of a point mutation in the second largest subunit of RNA polymerase I near the active center of the enzyme that results in an elongation-defective enzyme in the yeast Saccharomyces cerevisiae. In vivo, this mutant shows significant defects in rRNA processing and ribosome assembly. Taken together, these data suggest that transcription of rRNA by RNA polymerase I is linked to rRNA processing and maturation. Thus, RNA polymerase I, elongation factors, and rRNA sequence elements appear to function together to optimize transcription elongation, coordinating cotranscriptional interactions of many factors/snoRNAs with pre-rRNA for correct rRNA processing and ribosome assembly.

Expression of rRNA genes and nucleolus formation at ectopic chromosomal sites in the yeast Saccharomyces cerevisiae.

We constructed yeast strains in which rRNA gene repeats are integrated at ectopic sites in the presence or absence of the native nucleolus. At all three ectopic sites analyzed, near centromere CEN5, near the telomere of chromosome VI-R, and in middle of chromosome V-R (mid-V-R), a functional nucleolus was formed, and no difference in the expression of rRNA genes was observed. When two ribosomal DNA (rDNA) arrays are present, one native and the other ectopic, there is codominance in polymerase I (Pol I) transcription. We also examined the expression of a single rDNA repeat integrated into ectopic loci in strains with or without the native RDN1 locus. In a strain with reduced rRNA gene copies at RDN1 (approximately 40 copies), the expression of a single rRNA gene copy near the telomere was significantly reduced relative to the other ectopic sites, suggesting a less-efficient recruitment of the Pol I machinery from the RDN1 locus. In addition, we found a single rRNA gene at mid-V-R was as active as that within the 40-copy RDN1. Combined with the results of activity analysis of a single versus two tandem copies at CEN5, we conclude that tandem repetition is not required for efficient rRNA gene transcription.

Role of histone deacetylase Rpd3 in regulating rRNA gene transcription and nucleolar structure in yeast.

The 35S rRNA genes at the RDN1 locus in Saccharomyces cerevisiae can be transcribed by RNA polymerase (Pol) II in addition to Pol I, but Pol II transcription is usually silenced. The deletion of RRN9 encoding an essential subunit of the Pol I transcription factor, upstream activation factor, is known to abolish Pol I transcription and derepress Pol II transcription of rRNA genes, giving rise to polymerase switched (PSW) variants. We found that deletion of histone deacetylase gene RPD3 inhibits the appearance of PSW variants in rrn9 deletion mutants. This inhibition can be explained by the observed specific inhibition of Pol II transcription of rRNA genes by the rpd3Delta mutation. We propose that Rpd3 plays a role in the maintenance of an rRNA gene chromatin structure(s) that allows Pol II transcription of rRNA genes, which may explain the apparently paradoxical previous observation that rpd3 mutations increase, rather than decrease, silencing of reporter Pol II genes inserted in rRNA genes. We have additionally demonstrated that Rpd3 is not required for inhibition of Pol I transcription by rapamycin, supporting the model that Tor-dependent repression of the active form of rRNA genes during entry into stationary phase is Rpd3 independent.

Histones are required for transcription of yeast rRNA genes by RNA polymerase I.

Nucleosomes and their histone components have generally been recognized to act negatively on transcription. However, purified upstream activating factor (UAF), a transcription initiation factor required for RNA polymerase (Pol) I transcription in Saccharomyces cerevisiae, contains histones H3 and H4 and four nonhistone protein subunits. Other studies have shown that histones H3 and H4 are associated with actively transcribed rRNA genes. To examine their functional role in Pol I transcription, we constructed yeast strains in which synthesis of H3 is achieved from the glucose-repressible GAL10 promoter. We found that partial depletion of H3 (approximately 50% depletion) resulted in a strong inhibition (>80%) of Pol I transcription. A combination of biochemical analysis and electron microscopic (EM) analysis of Miller chromatin spreads indicated that initiation and elongation steps and rRNA processing were compromised upon histone depletion. A clear decrease in relative amounts of UAF, presumably caused by reduced stability, was also observed under the conditions of H3 depletion. Therefore, the observed inhibition of initiation can be explained, in part, by the decrease in UAF concentration. In addition, the EM results suggested that the defects in rRNA transcript elongation and processing may be a result of loss of histones from rRNA genes rather than (or in addition to) an indirect consequence of effects of histone depletion on expression of other genes. Thus, these results show functional importance of histones associated with actively transcribed rRNA genes.

RNA polymerase I remains intact without subunit exchange through multiple rounds of transcription in Saccharomyces cerevisiae.

Previous experiments using mammalian cells suggested that after each round of transcription, RNA polymerase I (Pol I) dissociates into subunits that leave and reenter the nucleolus as individual subunits, before formation of a new initiation complex. In this study, we show that the size and subunit composition of Pol I did not change significantly when Pol I was not engaged in rRNA transcription, brought about by either the absence of Pol I-specific rDNA template or specific inhibition of the transcription initiation step that requires Rrn3p. In fact, Pol I purified from cells completely lacking rDNA repeats was more active than when purified from wild-type cells in an in vitro transcription system designed to assay active Pol I-Rrn3p complexes. Furthermore, measurements of the exchange of A135 and A190 subunits between preexistent Pol I and newly synthesized Pol I showed that these two largest subunits of Pol I do not disassociate through many rounds of transcription in vivo. Thus, Pol I is not a dynamic protein complex but rather a stable enzyme.

SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast.

It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation. SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA. We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Delta and SIR2 strains. Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains. Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Delta relative to SIR2 strains. From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.

Tor pathway regulates Rrn3p-dependent recruitment of yeast RNA polymerase I to the promoter but does not participate in alteration of the number of active genes.

Yeast cells entering into stationary phase decrease rRNA synthesis rate by decreasing both the number of active genes and the transcription rate of individual active genes. Using chromatin immunoprecipitation assays, we found that the association of RNA polymerase I with the promoter and the coding region of rDNA is decreased in stationary phase, but association of transcription factor UAF with the promoter is unchanged. Similar changes were also observed when growing cells were treated with rapamycin, which is known to inhibit the Tor signaling system. Rapamycin treatment also caused a decrease in the amount of Rrn3p-polymerase I complex, similar to stationary phase. Because recruitment of Pol I to the rDNA promoter is Rrn3p-dependent as shown in this work, these data suggest that the decrease in the transcription rate of individual active genes in stationary phase is achieved by the Tor signaling system acting at the Rrn3p-dependent polymerase recruitment step. Miller chromatin spreads of cells treated with rapamycin and cells in post-log phase confirm this conclusion and demonstrate that the Tor system does not participate in alteration of the number of active genes observed for cells entering into stationary phase.

Silencing in yeast rDNA chromatin: reciprocal relationship in gene expression between RNA polymerase I and II.

About half of approximately 150 rRNA genes are transcriptionally active in Saccharomyces cerevisiae. Chromatin structures in the inactive, and not the active, copies were previously thought to silence both rRNA genes and reporter Pol II genes. Contrary to this belief, we found that silencing of reporters is much stronger in a mutant with approximately 25 rDNA copies, all of which are transcriptionally active. By integrating reporter gene mURA3 with an inactive rDNA copy missing the Pol I promoter, we found that mURA3 is not silenced in chromosomal rDNA repeats. Together with the demonstration of a requirement for active Pol I in silencing, these results show a reciprocal relationship in gene expression between Pol I and Pol II in rDNA.

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.

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.