Stimulation of bacteriophage T4 middle transcription by the T4 proteins MotA and AsiA occurs at two distinct steps in the transcription cycle

The bacteriophage T4 encodes proteins that are responsible for tightly regulating mRNA synthesis throughout phage development in Escherichia coli. The three classes of T4 promoters (early, middle, and late) are utilized sequentially by the host RNA polymerase as a result of phage-induced modifications. One such modification is the tight binding of the T4 AsiA protein to the σ70 subunit of the RNA polymerase. This interaction is pivotal for the transition between T4 early and middle transcription, since it both inhibits recognition of host and T4 early promoters and stimulates T4 middle mode synthesis. The activation of T4 middle transcription also requires the T4 MotA protein, bound specifically to its recognition sequence, the “Mot box,” which is centered at position −30 of these promoters. Accordingly, the two T4 proteins working in concert are sufficient to effectively switch the transcription specificity of the RNA polymerase holoenzyme. Herein, we investigate the mechanism of transcription activation and report that, while the presence of MotA and AsiA increases the initial recruitment of RNA polymerase to a T4 middle promoter, it does not alter the intrinsic stability of the discrete complexes formed. In addition, we have characterized the RNA polymerase-promoter species by UV laser footprinting and followed their evolution from open into initiating complexes. These data, combined with in vitro transcription assays, indicate that AsiA and MotA facilitate promoter escape, thereby stimulating the production of full-length transcripts.

The biochemical properties of the ATPase activity of a 70-kDa heat shock protein (Hsp70) are governed by the C-terminal domains

The cytosolic 70-kDa heat shock proteins (Hsp70s), Ssa and Ssb, of Saccharomyces cerevisiae are functionally distinct. Here we report that the ATPase activities of these two classes of Hsp70s exhibit different kinetic properties. The Ssa ATPase has properties similar to those of other Hsp70s studied, such as DnaK and Hsc70. Ssb, however, has an unusually low steady-state affinity for ATP but a higher maximal velocity. In addition, the ATPase activity of Hsp70s, like that of Ssa1, depends on the addition of K+ whereas Ssb activity does not. Suprisingly, the isolated 44-kDa ATPase domain of Ssb has a Km and Vmax for ATP hydrolysis similar to those of Ssa, rather than those of full length Ssb. Analysis of Ssa/Ssb fusion proteins demonstrates that the Ssb peptide-binding domain fused to the Ssa ATPase domain generates an ATPase of relatively high activity and low steady-state affinity for ATP similar to that of native Ssb. Therefore, at least some of the biochemical differences between the ATPases of these two classes of Hsp70s are not intrinsic to the ATPase domain itself. The differential influence of the peptide-binding domain on the ATPase domain may, in part, explain the functional uniqueness of these two classes of Hsp70s.

Formation of the preprimosome protects λ O from RNA transcription-dependent proteolysis by ClpP/ClpX

Using the bacteriophage λ DNA replication system, composed entirely of purified proteins, we have tested the accessibility of the short-lived λ O protein to the ClpP/ClpX protease during the various stages of λ DNA replication. We find that binding of λ O protein to its oriλ DNA sequence, leading to the so-called “O-some” formation, largely inhibits its degradation. On the contrary, under conditions permissive for transcription, the λ O protein bound to the oriλ sequence becomes largely accessible to ClpP/ClpX-mediated proteolysis. However, when the λ O protein is part of the larger oriλ:O⋅P⋅DnaB preprimosomal complex, transcription does not significantly increase ClpP/ClpX-dependent λ O degradation. These results show that transcription can stimulate proteolysis of a protein that is required for the initiation of DNA replication.

Differential regulation of transcription: Repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins

Kss1, a yeast mitogen-activated protein kinase (MAPK), in its unphosphorylated (unactivated) state binds directly to and represses Ste12, a transcription factor necessary for expression of genes whose promoters contain filamentous response elements (FREs) and genes whose promoters contain pheromone response elements (PREs). Herein we show that two nuclear proteins, Dig1 and Dig2, are required cofactors in Kss1-imposed repression. Dig1 and Dig2 cooperate with Kss1 to repress Ste12 action at FREs and regulate invasive growth in a naturally invasive strain. Kss1-imposed Dig-dependent repression of Ste12 also occurs at PREs. However, maintenance of repression at PREs is more dependent on Dig1 and/or Dig2 and less dependent on Kss1 than repression at FREs. In addition, derepression at PREs is more dependent on MAPK-mediated phosphorylation than is derepression at FREs. Differential utilization of two types of MAPK-mediated regulation (binding-imposed repression and phosphorylation-dependent activation), in combination with distinct Ste12-containing complexes, contributes to the mechanisms by which separate extracellular stimuli that use the same MAPK cascade can elicit two different transcriptional responses.

Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I

RNA polymerase I (Pol I) transcription in the yeast Saccharomyces cerevisiae is greatly stimulated in vivo and in vitro by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter, such that once bound (in vitro), UAF does not readily exchange onto a competing template. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription in vivo: RRN5, RRN9, and RRN10. Two others, p30 and p18, have remained uncharacterized. We report here that the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 shows that it is histone H3. In addition, histone H4 was found in UAF, and myc-tagged histone H4 could be used to affinity-purify UAF. Histones H2A and H2B were not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I.

Glucocorticoid-mediated repression of nuclear factor-κBdependent transcription involves direct interference with transactivation

Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-κB. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IκB molecule, which in turn would bind and remove activated, DNA-bound NF-κB complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-κB-dependent transcription, without changing the expression level of IκB. Neither the mRNA of IκB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-κB also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-κB. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-κB p65 subunit.

Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates

Several models have been proposed for the mechanism of transcript termination by Escherichia coli RNA polymerase at rho-independent terminators. Yager and von Hippel (Yager, T. D. & von Hippel, P. H. (1991) Biochemistry 30, 1097–118) postulated that the transcription complex is stabilized by enzyme–nucleic acid interactions and the favorable free energy of a 12-bp RNA–DNA hybrid but is destabilized by the free energy required to maintain an extended transcription bubble. Termination, by their model, is viewed simply as displacement of the RNA transcript from the hybrid helix by reformation of the DNA helix. We have proposed an alternative model where the RNA transcript is stably bound to RNA polymerase primarily through interactions with two single-strand specific RNA-binding sites; termination is triggered by formation of an RNA hairpin that reduces binding of the RNA to one RNA-binding site and, ultimately, leads to its ejection from the complex. To distinguish between these models, we have tested whether E. coli RNA polymerase can terminate transcription at rho-independent terminators on single-stranded DNA. RNA polymerase cannot form a transcription bubble on these templates; thus, the Yager–von Hippel model predicts that intrinsic termination will not occur. We find that transcript elongation on single-stranded DNA templates is hindered somewhat by DNA secondary structure. However, E. coli RNA polymerase efficiently terminates and releases transcripts at several rho-independent terminators on such templates at the same positions as termination occurs on duplex DNAs. Therefore, neither the nontranscribed DNA strand nor the transcription bubble is essential for rho-independent termination by E. coli RNA polymerase.

Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein α gene

During adipocyte differentiation, the expression of C/EBPα is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPα gene led to the identification of a potential repressive element within the proximal 5′ flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPα undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPα promoter-luciferase constructs containing both the proximal 5′ flanking and the entire 5′ untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPα gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5′ untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5′ untranslated region. Although mutation of either CUP element in constructs containing both the 5′ flanking and 5′ untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPα gene prior to induction of the adipocyte differentiation program.

Methylation of the minimal promoter of an embryonic globin gene silences transcription in primary erythroid cells

Methylation of cytosines in the dinucleotide CpG has been shown to suppress transcription of a number of tissue-specific genes, yet the precise mechanism is not fully understood. The vertebrate globin genes were among the first examples in which an inverse correlation was shown between CpG methylation and transcription. We studied the methylation pattern of the 235-bp ρ-globin gene promoter in genomic DNA from primary chicken erythroid cells using the sodium bisulfite conversion technique and found all CpGs in the promoter to be methylated in erythroid cells from adult chickens in which the ρ-globin gene is silent but unmethylated in 5-day (primitive) embryonic red cells in which the gene is transcribed. To elucidate further the mechanism of methylation-induced silencing, an expression construct consisting of 235 bp of 5′ promoter sequence of the ρ-globin gene along with a strong 5′ erythroid enhancer driving a chloramphenicol acetyltransferase reporter gene, ρ-CAT, was transfected into primary avian erythroid cells derived from 5-day embryos. Methylation of just the 235-bp ρ-globin gene promoter fragment at every CpG resulted in a 20- to 30-fold inhibition of transcription, and this effect was not overridden by the presence of potent erythroid-specific enhancers. The ability of the 235-bp ρ-globin gene promoter to bind to a DNA Methyl Cytosine binding Protein Complex (MeCPC) was tested in electrophoretic mobility shift assays utilizing primary avian erythroid cell nuclear extract. The results were that fully methylated but not unmethylated 235-bp ρ-globin gene promoter fragment could compete efficiently for MeCPC binding. These results are a direct demonstration that site-specific methylation of a globin gene promoter at the exact CpGs that are methylated in vivo can silence transcription in homologous primary erythroid cells. Further, these data implicate binding of MeCPC to the promoter in the mechanism of silencing.

Transcription factor Mts1/Mts2 (Atf1/Pcr1, Gad7/Pcr1) activates the M26 meiotic recombination hotspot in Schizosaccharomyces pombe

Homologous recombination hotspots increase the frequency of recombination in nearby DNA. The M26 hotspot in the ade6 gene of Schizosaccharomyces pombe is a meiotic hotspot with a discrete, cis-acting nucleotide sequence (5′-ATGACGT-3′) defined by extensive mutagenesis. A heterodimeric M26 DNA binding protein, composed of subunits Mts1 and Mts2, has been identified and purified 40,000-fold. Cloning, disruption, and genetic analyses of the mts genes demonstrate that the Mts1/Mts2 heterodimer is essential for hotspot activity. This provides direct evidence that a specific trans-acting factor, binding to a cis-acting site with a unique nucleotide sequence, is required to activate this meiotic hotspot. Intriguingly, the Mts1/Mts2 protein subunits are identical to the recently described transcription factors Atf1 (Gad7) and Pcr1, which are required for a variety of stress responses. However, we report differential dependence on the Mts proteins for hotspot activation and stress response, suggesting that these proteins are multifunctional and have distinct activities. Furthermore, ade6 mRNA levels are equivalent in hotspot and nonhotspot meioses and do not change in mts mutants, indicating that hotspot activation is not a consequence of elevated transcription levels. These findings suggest an intimate but separable link between the regulation of transcription and meiotic recombination. Other studies have recently shown that the Mts1/Mts2 protein and M26 sites are involved in meiotic recombination elsewhere in the S. pombe genome, suggesting that these factors help regulate the timing and distribution of homologous recombination.

Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants mab1–1, mab2–1, and mab3–1 (for maintenance of BMV functions), were selected for initial study. Each of these mutants has a single, recessive, chromosomal mutation that inhibits accumulation of positive- and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the RNA replication template was introduced by in vivo transcription from DNA or by transfection of yeast with in vitro transcripts, confirming that cytoplasmic RNA replication steps were defective. mab1–1, mab2–1, and mab3–1 slowed yeast growth to varying degrees and were temperature-sensitive, showing that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the accumulation of 2a mRNA and the polymerase-like 2a protein, revealing a new level of viral regulation. In association with their other effects, mab1–1 and mab2–1 blocked the ability of 1a to stimulate 2a mRNA and protein accumulation, whereas mab3–1 had elevated 2a protein accumulation. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression and accumulation of 2a.

Selection of peptides that functionally replace a zinc finger in the Sp1 transcription factor by using a yeast combinatorial library

We have developed a strategy for the identification of peptides able to functionally replace a zinc finger domain in a transcription factor. This strategy could have important ramifications for basic research on gene regulation and for the development of therapeutic agents. In this study in yeast, we expressed chimeric proteins that included a random peptide combinatorial library in association with two zinc finger domains and a transactivating domain. The library was screened for chimeric proteins capable of activating transcription from a target sequence in the upstream regulatory regions of selectable or reporter genes. In a screen of approximately 1.5 × 107 transformants we identified 30 chimeric proteins that exhibited transcriptional activation, some of which were able to discriminate between wild-type and mutant DNA targets. Chimeric library proteins expressed as glutathione S-transferase fusions bound to double-stranded oligonucleotides containing the target sequence, suggesting that the chimeras bind directly to DNA. Surprisingly, none of the peptides identified resembled a zinc finger or other well-known transcription factor DNA binding domain.

Ash1p is a site-specific DNA-binding protein that actively represses transcription

ASH1 encodes a protein that is localized specifically to the daughter cell nucleus, where it has been proposed to repress transcription of the HO gene. Using Ash1p purified from baculovirus-infected insect cells, we have shown that Ash1p binds specific DNA sequences in the HO promoter. DNase I protection analyses showed that Ash1p recognizes a consensus sequence, YTGAT. Mutation of this consensus abolishes Ash1p DNA binding in vitro. We have shown that Ash1p requires an intact zinc-binding domain in its C terminus for repression of HO in vivo and that this domain may be involved in DNA binding. A heterologous DNA-binding domain fused to an N-terminal segment of Ash1p functions as an active repressor of transcription. Our studies indicate that Ash1p is a DNA-binding protein of the GATA family with a separable transcriptional repression domain.

The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex

The E2F transcription factors play a key role in the regulation of cellular proliferation and terminal differentiation. E2F6 is the most recently identified and the least well understood member of the E2F family. It is only distantly related to the other E2Fs and lacks the sequences responsible for both transactivation and binding to the retinoblastoma protein. Consistent with this finding, E2F6 can behave as a dominant negative inhibitor of the other E2F family members. In this study, we continue to investigate the possible role(s) of E2F6 in vivo. We report the isolation of RYBP, a recently identified member of the mammalian polycomb complex, as an E2F6-interacting protein. Mapping studies indicate that RYBP binds within the known “repression domain” of E2F6. Moreover, we demonstrate that endogenous E2F6 and polycomb group proteins, including RYBP, Ring1, MEL-18, mph1, and the oncoprotein Bmi1, associate with one another. These findings suggest that the biological properties of E2F6 are mediated through its ability to recruit the polycomb transcriptional repressor complex.

The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

During retinogenesis, the Xenopus basic helix–loop–helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.