Microbial metallothioneins.

Bacterial metallothioneins bind, sequester and buffer excess intracellular zinc. At present, the vast majority of the available experimental data relate to cyanobacterial metallothionein, SmtA, from Synechococcus PCC 7942. SmtA is required for normal resistance to zinc and smtA-mediated zinc resistance has been used as a selectable marker. The imidazole groups of histidine residues, in addition to the thiol groups of cysteine residues, co-ordinate zinc in bacterial metallothioneins. The structure of bacterial metallothionein must facilitate some discrimination between 'adventitious' and 'adventageous' zinc-binding sites such that under excess zinc conditions metal is predominantly scavenged from the former. It remains unclear whether or not bacterial metallothionein also acts as a zinc store that supplies zinc-requiring proteins or if under some conditions it deactivates a subset of proteins via zinc removal. Expression of smtA is induced in response to elevated concentrations of zinc via the action of SmtB. SmtB has some sequence similarity to the arsenic responsive repressor ArsR and genes encoding related proteins are present in many bacterial genomes. Metal perception by SmtB differs from ArsR. The latter contains a characteristic Cys-Val-Cys motif associated with a DNA-binding helix-turn-helix (the ArsR motif), while the former contains metal-binding motifs associated with a carboxyl-terminal alpha-helix that forms the interface between SmtB dimers (the SmtB motif). Some SmtB-ArsR family proteins, including the zinc sensor ZiaR from the cyanobacterium Synechocystis PCC 6803, have the metal-sensory motifs of both SmtB and ArsR. The mechanisms of action, and the features that allow discrimination between different metal ions by these sensors, are discussed.

Transcriptional regulators of the Schizosaccharomyces pombe fbp1 gene include two redundant Tup1p-like corepressors and the CCAAT binding factor activation complex.

The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

The symmetry of the yeast U6 RNA gene's TATA box and the orientation of the TATA-binding protein in yeast TFIIIB.

The central RNA polymerase III (Pol III) transcription factor TFIIIB is composed of the TATA-binding protein (TBP), Brf, a protein related to TFIIB, and the product of the newly cloned TFC5 gene. TFIIIB assembles autonomously on the upstream promoter of the yeast U6 snRNA (SNR6) gene in vitro, through the interaction of its TBP subunit with a consensus TATA box located at base pair -30. As both the DNA-binding domain of TBP and the U6 TATA box are nearly twofold symmetrical, we have examined how the binding polarity of TFIIIB is determined. We find that TFIIIB can bind to the U6 promoter in both directions, that TBP is unable to discern the natural polarity of the TATA element and that, as a consequence, the U6 TATA box is functionally symmetrical. A modest preference for TFIIIB binding in the natural direction of the U6 promoter is instead dictated by flanking DNA. Because the assembly of TFIIIB on the yeast U6 gene in vivo occurs via a TFIIIC-dependent mechanism, we investigated the influence of TFIIIC on the binding polarity of TFIIIB. TFIIIC places TFIIIB on the promoter in one direction only; thus, it is TFIIIC that primarily specifies the direction of transcription. Experiments using TFIIIB reconstituted with the altered DNA specificity mutant TBPm3 demonstrate that in the TFIIIB-U6 promoter complex, the carboxy-terminal repeat of TBP contacts the upstream half of the TATA box. This orientation of yeast TBP in Pol III promoter-bound TFIIIB is the same as in Pol II promoter-bound TFIID and in TBP-DNA complexes that have been analyzed by X-ray crystallography.

TFIIIB placement on a yeast U6 RNA gene in vivo is directed primarily by TFIIIC rather than by sequence-specific DNA contacts.

The Saccharomyces cerevisiae U6 RNA gene (SNR6), which is transcribed by RNA polymerase III, has an unusual combination of promoter elements: an upstream TATA box, an intragenic A block, and a downstream B block. In tRNA genes, the A and B blocks are binding sites for the transcription initiation factor TFIIIC, which positions TFIIIB a fixed distance upstream of the A block. However, in vitro transcription of SNR6 with purified components requires neither TFIIIC nor the A and B blocks, presumably because TFIIIB recognizes the upstream sequences directly. Here we demonstrate that TFIIIB placement on SNR6 in vivo is directed primarily by the TFIIIC-binding elements rather than by upstream sequences. We show that the A block is a stronger start site determinant than the upstream sequences when the two are uncoupled by an insertion mutation. Furthermore, while TFIIIC-independent in vitro transcription of SNR6 is highly sensitive to TATA box point mutations, in vivo initiation on SNR6 is only marginally sensitive to such mutations unless the A block is mutated. Intriguingly, a deletion downstream of the U6 RNA coding region that reduces A-to-B block spacing also increases in vivo dependence on the TATA box. Moreover, this deletion results in the appearance of micrococcal nuclease-hypersensitive sites in the TFIIIB chromatin footprint, indicating that TFIIIB binding is disrupted by a mutation 150 bp distant. This and additional chromatin footprinting data suggest that SNR6 is assembled into a nucleoprotein complex that facilitates the TFIIIC-dependent binding of TFIIIB.

Hydrolytic cleavage of nascent RNA in RNA polymerase III ternary transcription complexes.

Highly purified yeast RNA polymerase III ternary complexes were found to possess a hydrolytic chain retracting activity that cleaves nascent RNA from its 3'-OH end. Most of the shortened transcripts were capable of resuming RNA chain elongation, indicating that they remain stably associated with the enzyme-DNA complex. Analysis of the products of cleavage indicated that retraction primarily occurred in dinucleotide increments, but that mononucleotides were also excised at lower frequency. The ribonuclease activity was totally dependent on the presence of a divalent cation and was stimulated by the addition of non-cognate ribonucleotides. The inclusion of ATP in the reaction enhanced both the rate and extent of transcript cleavage. Evidence suggesting that the hydrolytic activity is intrinsic to RNA polymerase III and factor-independent is also presented. Transcript cleavage by RNA polymerase III ternary complexes appears to be more closely related to the intrinsic nucleolytic activity of vaccinia virus RNA polymerase ternary complexes than to TFIIS-dependent cleavage that has been described for RNA polymerase II ternary complexes.