Transcriptional elements involved in the repression of ribosomal protein synthesis.

The ribosomal proteins (RPs) of Saccharomyces cerevisiae are encoded by 137 genes that are among the most transcriptionally active in the genome. These genes are coordinately regulated: a shift up in temperature leads to a rapid, but temporary, decline in RP mRNA levels. A defect in any part of the secretory pathway leads to greatly reduced ribosome synthesis, including the rapid loss of RP mRNA. Here we demonstrate that the loss of RP mRNA is due to the rapid transcriptional silencing of the RP genes, coupled to the naturally short lifetime of their transcripts. The data suggest further that a global inhibition of polymerase II transcription leads to overestimates of the stability of individual mRNAs. The transcription of most RP genes is activated by two Rap1p binding sites, 250 to 400 bp upstream from the initiation of transcription. Rap1p is both an activator and a silencer of transcription. The swapping of promoters between RPL30 and ACT1 or GAL1 demonstrated that the Rap1p binding sites of RPL30 are sufficient to silence the transcription of ACT1 in response to a defect in the secretory pathway. Sir3p and Sir4p, implicated in the Rap1p-mediated repression of silent mating type genes and of telomere-proximal genes, do not influence such silencing of RP genes. Sir2p, implicated in the silencing both of the silent mating type genes and of genes within the ribosomal DNA locus, does not influence the repression of either RP or rRNA genes. Surprisingly, the 180-bp sequence of RPL30 that lies between the Rap1p sites and the transcription initiation site is also sufficient to silence the Gal4p-driven transcription in response to a defect in the secretory pathway, by a mechanism that requires the silencing region of Rap1p. We conclude that for Rap1p to activate the transcription of an RP gene it must bind to upstream sequences; yet for Rap1p to repress the transcription of an RP gene it need not bind to the gene directly. Thus, the cell has evolved a two-pronged approach to effect the rapid extinction of RP synthesis in response to the stress imposed by a heat shock or by a failure of the secretory pathway. Calculations based on recent transcriptome data and on the half-life of the RP mRNAs suggest that in a rapidly growing cell the transcription of RP mRNAs accounts for nearly 50% of the total transcriptional events initiated by RNA polymerase II. Thus, the sudden silencing of the RP genes must have a dramatic effect on the overall transcriptional economy of the cell.

Protein kinase C enables the regulatory circuit that connects membrane synthesis to ribosome synthesis in Saccharomyces cerevisiae.

The balanced growth of a cell requires the integration of major systems such as DNA replication, membrane biosynthesis, and ribosome formation. An example of such integration is evident from our recent finding that, in Saccharomyces cerevisiae, any failure in the secretory pathway leads to severe repression of transcription of both rRNA and ribosomal protein genes. We have attempted to determine the regulatory circuit(s) that connects the secretory pathway with the transcription of ribosomal genes. Experiments show that repression does not occur through the circuit that responds to misfolded proteins in the endoplasmic reticulum, nor does it occur through circuits known to regulate ribosome synthesis, e.g. the stringent response, or the cAMP pathway. Rather, it appears to depend on a stress response at the plasma membrane that is transduced through protein kinase C (PKC). Deletion of PKC1 relieves the repression of both ribosomal protein and rRNA genes that occurs in response to a defect in the secretory pathway. We propose that failure of the secretory pathway prevents the synthesis of new plasma membrane. As protein synthesis continues, stress develops in the plasma membrane. This stress is monitored by Pkc1p, which initiates a signal transduction pathway that leads to repression of transcription of the rRNA and ribosomal protein genes. The importance of the transcription of the 137 ribosomal protein genes to the economy of the cell is apparent from the existence of at least three distinct pathways that can effect the repression of this set of genes.

Does Saccharomyces need an organized nucleolus?

The ribosomal RNA (rRNA) genes of most eukaryotic organisms are arranged in one or more tandem arrays, often termed nucleolar organizer regions. The biological implications of this tandem organization are not known. We have tested the requirement for such a chromosomal organization by directly comparing the transcription and processing of rRNA in isogenic strains of Saccharomyces cerevisiae that differ only in the organization of their rRNA genes. Strain L-1489 carries the RDN locus, consisting of 100-150 copies of the rRNA genes in a tandem array on chromosome XII. Strain L-1521 has a complete deletion of the RDN array, but carries many copies of a plasmid that includes a single rRNA gene. While this strain grows reasonably well, the average transcriptional activity of the plasmid genes is substantially less than that of the chromosomal copies. However, there is little difference in the processing of the 35S pre-rRNA to the mature 25S:5.8S and 18S products. Thus, neither a chromosomal location nor a tandem repeat of the rRNA genes is important for the assembly and function of the many protein and RNA molecules necessary for the processing of the rRNA transcripts. We investigated the consequence of a dispersed gene arrangement on nucleolar structure. Immunofluorescence microscopy revealed that in strain L-1521 the yeast fibrillarin, Nop1p, rather than being confined to a defined nucleolus at the edge of the nucleus as it is in cells with the normal arrangement of rRNA genes, is spread throughout the nucleus. This observation implies that each plasmid rRNA gene can serve as a nucleolar organizer. Finally, data from pulse-labeling experiments show that the repression of rRNA transcription due to failure of the secretory pathway is independent of whether the rRNA genes are at the RDN locus on chromosome XII or on plasmids. This result suggests that the regulation of rRNA transcription occurs at the level of soluble factors rather than chromatin structure.

The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae.

The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C-terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon-specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA-containing strains produced an antisuppressor phenotype. These data are consistent with Sup35p and Sup45p forming a complex with release factor properties. Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti-suppression only if that strain was also overexpressing the yeast SUP35 gene. Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors. We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C-terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction.

The dominant PNM2- mutation which eliminates the psi factor of Saccharomyces cerevisiae is the result of a missense mutation in the SUP35 gene.

The PNM2- mutation of Saccharomyces cerevisiae eliminates the extrachromosomal element psi. PNM2 is closely linked to the omnipotent suppressor gene SUP35 (also previously identified as SUP2, SUF12, SAL3 and GST1). We cloned PNM2- and showed that PNM2 and SUP35 are the same gene. We sequenced the PNM2- mutant allele and found a single G-->A transition within the N-terminal domain of the protein. We tested the effects of various constructs of SUP35 and PNM2- on psi inheritance and on allosuppressor and antisuppressor functions of the gene. We found that the C-terminal domain of SUP35 protein (SUP35p) could be independently expressed; expression produced dominant antisuppression. Disruption of the N-terminal domain of PNM2- destroyed the ability to eliminate psi. These results imply that the domains of SUP35p act in an antagonistic manner: the N-terminal domain decreases chain-termination fidelity, while the C-terminal domain imposes fidelity. Two transcripts were observed for SUP35, a major band at 2.4 kb and a minor band at 1.3 kb; the minor band corresponds to 3' sequences only. We propose a model for the function of SUP35, in which comparative levels of N- and C-terminal domains of SUP35p at the ribosome modulate translation fidelity.

Expression and inheritance of the yeast extrachromosomal element psi do not depend on RNA polymerase I.

The extrachromosomal element psi affects translation fidelity in the yeast Saccharomyces cerevisiae by increasing the efficiency of tRNA-mediated ochre suppression. The nature of the psi factor is unknown, although there is evidence that 3-microns circles from psi+ strains can be used to transform psi- cells to psi+. The 3-microns circles are extrachromosomal copies of the repeating ribosomal DNA unit, which is organized into two transcription units: the 35s rRNA precursor transcribed by RNA polymerase I, and the 5s rRNA transcribed by RNA polymerase III. We used a strain containing a mutation in RNA polymerase I to test whether psi expression and inheritance depended on RNA polI. Neither expression nor inheritance of psi requires intact RNA polI.

Identification of Escherichia coli ribosomal proteins by an alternative two-dimensional electrophoresis system.

We have developed a two-dimensional gel electrophoretic system for the identification of Escherichia coli ribosomal proteins that involves the use of acid-urea in the first dimension and sodium dodecyl sulfate in the second dimension. This system has high sensitivity, resolution, and speed, and it is more convenient than others previously described. We have identified individual E. coli ribosomal proteins by this system.

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins.

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.