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This file contain Supplementary Information that is relevant to the main body of the text. Supplementary Figure S1. Supplementary Figure S2. Half-lives of WT and mutant I332fs*14 p53 in U2OS and HCT116 cells. Supplementary Figure S3. Subcellular localization of p53 FS mutants expressed in U2OS and HCT116 cells. Supplementary Figure S4. Cross-linked species detected in cells grown in the absence or presence of Photo-Met. Supplementary Figure S5. Transcriptional activity of p53 FS mutants in U2OS and HCT116 cells. Supplementary Figure S6. Transactivation potential of N345K* and N345fs*26 mutants in U2OS cells. Supplementary Figure S7. Cell cycle profiles of I332fs*14-expressing HCT116 and U2OS cells. Supplementary Figure S8. Pro-apoptotic activity of I332fs*14 p53.

p53 mutations that result in loss of transcriptional activity are commonly found in numerous types of cancer. While the majority of these are missense mutations that map within the central DNA-binding domain, truncations and/or frameshift mutations can also occur due to various nucleotide substitutions, insertions, or deletions. These changes result in mRNAs containing premature stop codons that are translated into a diverse group of C-terminally truncated proteins. Here we characterized three p53 frameshift mutant proteins expressed from the endogenous TP53 locus in U2OS osteosarcoma and HCT116 colorectal cancer cell lines. These mutants retain intact DNA-binding domains but display altered oligomerization properties. Despite their abnormally high expression levels, they are mostly transcriptionally inactive and unable to initiate a stimuli-induced transcriptional program characteristic of wild-type p53. However, one of these variant p53 proteins, I332fs*14, which resembles naturally expressed TAp53 isoforms β and γ, retains some residual antiproliferative activity and can induce cellular senescence in HCT116 cells. Cells expressing this mutant also display decreased motility in migration assays. Hence, this p53 variant exhibits a combination of loss-of-gain and gain-of-function characteristics, distinguishing it from both wild type p53 and p53 loss.Implications:p53 frameshift mutants display a mixture of residual antiproliferative and neomorphic functions that may be differentially exploited for targeted therapy.

p53 mutations that result in loss of transcriptional activity are commonly found in numerous types of cancer. While the majority of these are missense mutations that map within the central DNA-binding domain, truncations and/or frameshift mutations can also occur due to various nucleotide substitutions, insertions, or deletions. These changes result in mRNAs containing premature stop codons that are translated into a diverse group of C-terminally truncated proteins. Here we characterized three p53 frameshift mutant proteins expressed from the endogenous TP53 locus in U2OS osteosarcoma and HCT116 colorectal cancer cell lines. These mutants retain intact DNA-binding domains but display altered oligomerization properties. Despite their abnormally high expression levels, they are mostly transcriptionally inactive and unable to initiate a stimuli-induced transcriptional program characteristic of wild-type p53. However, one of these variant p53 proteins, I332fs*14, which resembles naturally expressed TAp53 isoforms β and γ, retains some residual antiproliferative activity and can induce cellular senescence in HCT116 cells. Cells expressing this mutant also display decreased motility in migration assays. Hence, this p53 variant exhibits a combination of loss-of-gain and gain-of-function characteristics, distinguishing it from both wild type p53 and p53 loss. Implications: p53 frameshift mutants display a mixture of residual antiproliferative and neomorphic functions that may be differentially exploited for targeted therapy.

Significance One-tenth of human genes produce proteins called transcription factors (TFs) that bind to our genome and read the local DNA sequence. They work together to regulate the degree to which each gene is expressed. The affinity with which DNA is bound by a particular TF can vary more than a thousand-fold with different DNA sequences. This study presents the first computational method able to quantify the sequence-affinity relationship almost perfectly over the full affinity range. It achieves this by analyzing data from experiments that use massively parallel DNA sequencing to comprehensively probe protein–DNA interactions. Strikingly, it can accurately predict the effect in vivo of DNA mutations on gene expression levels in fly embryos even for very-low-affinity binding sites., Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Although DNA modifications play an important role in gene regulation, the underlying mechanisms remain elusive. We developed EpiSELEX-seq to probe the sensitivity of transcription factor binding to DNA modification in vitro using massively parallel sequencing. Feature-based modeling quantifies the effect of cytosine methylation (5mC) on binding free energy in a position-specific manner. Application to the human bZIP proteins ATF4 and C/EBPβ and three different Pbx-Hox complexes shows that 5mCpG can both increase and decrease affinity, depending on where the modification occurs within the protein-DNA interface. The TF paralogs tested vary in their methylation sensitivity, for which we provide a structural rationale. We show that 5mCpG can also enhance in vitro p53 binding and provide evidence for increased in vivo p53 occupancy at methylated binding sites, correlating with primed enhancer histone marks. Our results establish a powerful strategy for dissecting the epigenomic modulation of protein-DNA interactions and their role in gene regulation.

The p53 tumor suppressor is a transcription factor (TF) that exerts antitumor functions through its ability to regulate the expression of multiple genes. Within the p53 protein resides a relatively short unstructured C-terminal domain (CTD) that remarkably participates in virtually every aspect of p53 performance as a TF. Because these aspects are often interdependent and it is not always possible to dissect them experimentally, there has been a great deal of controversy about the CTD. In this review we evaluate the significance and key features of this interesting region of p53 and its impact on the many aspects of p53 function in light of previous and more recent findings.

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

DNA binding by numerous transcription factors including the p53 tumor suppressor protein constitutes a vital early step in transcriptional activation. While the role of the central core DNA binding domain (DBD) of p53 in site-specific DNA binding has been established, the contribution of the sequence-independent C-terminal domain (CTD) is still not well understood. We investigated the DNA-binding properties of a series of p53 CTD variants using a combination of in vitro biochemical analyses and in vivo binding experiments. Our results provide several unanticipated and interconnected findings. First, the CTD enables DNA binding in a sequence-dependent manner that is drastically altered by either its modification or deletion. Second, dependence on the CTD correlates with the extent to which the p53 binding site deviates from the canonical consensus sequence. Finally, the CTD enables stable formation of p53-DNA complexes to divergent binding sites via DNA-induced conformational changes within the DBD itself.

The p53 tumor suppressor protein is the most well studied as a regulator of transcription in the nucleus, where it exists primarily as a tetramer. However, there are other oligomeric states of p53 that are relevant to its regulation and activities. In unstressed cells, p53 is normally held in check by MDM2 that targets p53 for transcriptional repression, proteasomal degradation, and cytoplasmic localization. Here we discovered a hydrophobic region within the MDM2 N-terminal domain that binds exclusively to the dimeric form of the p53 C-terminal domain in vitro. In cell-based assays, MDM2 exhibits superior binding to, hyperdegradation of, and increased nuclear exclusion of dimeric p53 when compared with tetrameric wild-type p53. Correspondingly, impairing the hydrophobicity of the newly identified N-terminal MDM2 region leads to p53 stabilization. Interestingly, we found that dimeric mutant p53 is partially unfolded and is a target for ubiquitin-independent degradation by the 20S proteasome. Finally, forcing certain tumor-derived mutant forms of p53 into dimer configuration results in hyperdegradation of mutant p53 and inhibition of p53-mediated cancer cell migration. Gaining insight into different oligomeric forms of p53 may provide novel approaches to cancer therapy.

Long understood as a bona fide tumor suppressor that safeguards the integrity of the genome via regulating numerous cellular outcomes, p53 may also exert its decisive and versatile functions by controlling DNA methylation. In this issue of Genes & Development, Tovy and colleagues (pp. 959–972) report that, in naïve mouse embryonic stem cells (ESCs), p53 controls DNA methylation homeostasis by regulating the expression of key counteracting components of the DNA methylation machinery. Their findings indicate that p53 may exert its “guardian of genome” duties at least in part via safeguarding the epigenome of ESCs.

Monocytic leukemia zinc finger (MOZ)/KAT6A is a MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST)-type histone acetyltransferase that functions as a coactivator for acute myeloid leukemia 1 protein (AML1)- and Ets family transcription factor PU.1-dependent transcription. We previously reported that MOZ directly interacts with p53 and is essential for p53-dependent selective regulation of p21 expression. We show here that MOZ is an acetyltransferase of p53 at K120 and K382 and colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress. The MOZ–PML–p53 interaction enhances MOZ-mediated acetylation of p53, and this ternary complex enhances p53-dependent p21 expression. Moreover, we identified an Akt/protein kinase B recognition sequence in the PML-binding domain of MOZ protein. Akt-mediated phosphorylation of MOZ at T369 has a negative effect on complex formation between PML and MOZ. As a result of PML-mediated suppression of Akt, the increased PML–MOZ interaction enhances p21 expression and induces p53-dependent premature senescence upon forced PML expression. Our research demonstrates that MOZ controls p53 acetylation and transcriptional activity via association with PML.

Functional in a tetrameric state, the protein product of the p53 tumor suppressor gene confers its tumor-suppressive activity by transactivating genes which promote cell-cycle arrest, senescence, or programmed cell death. How p53 distinguishes between these divergent outcomes is still a matter of considerable interest. Here we discuss the impact of 2 mutations in the tetramerization domain that confer unique properties onto p53. By changing lysines 351 and 357 to arginine, thereby blocking all post-translational modifications of these residues, DNA binding and transcriptional regulation by p53 remain virtually unchanged. On the other hand, by changing these lysines to glutamine (2KQ-p53), thereby neutralizing their positive charge and potentially mimicking acetylation, p53 is impaired in the induction of cell cycle arrest and yet can still effectively induce cell death. Surprisingly, when 2KQ-p53 is expressed at high levels in H1299 cells, it can bind to and transactivate numerous p53 target genes including p21, but not others such as miR-34a and cyclin G1 to the same extent as wild-type p53. Our findings show that strong induction of p21 is not sufficient to block H1299 cells in G1, and imply that modification of one or both of the lysines within the tetramerization domain may serve as a mechanism to shunt p53 from inducing cell cycle arrest.

Motivation: Motif discovery is now routinely used in high-throughput studies including large-scale sequencing and proteomics. These datasets present new challenges. The first is speed. Many motif discovery methods do not scale well to large datasets. Another issue is identifying discriminative rather than generative motifs. Such discriminative motifs are important for identifying co-factors and for explaining changes in behavior between different conditions. Results: To address these issues we developed a method for DECOnvolved Discriminative motif discovery (DECOD). DECOD uses a k-mer count table and so its running time is independent of the size of the input set. By deconvolving the k-mers DECOD considers context information without using the sequences directly. DECOD outperforms previous methods both in speed and in accuracy when using simulated and real biological benchmark data. We performed new binding experiments for p53 mutants and used DECOD to identify p53 co-factors, suggesting new mechanisms for p53 activation. Availability: The source code and binaries for DECOD are available at http://www.sb.cs.cmu.edu/DECOD Contact: zivbj@cs.cmu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

The p53 tumor suppressor interacts with its negative regulator Mdm2 via the former’s N-terminal region and core domain. Yet the extreme p53 C-terminal region contains lysine residues ubiquitinated by Mdm2 and can bear post-translational modifications that inhibit Mdm2–p53 association. We show that, the Mdm2–p53 interaction is decreased upon deletion, mutation or acetylation of the p53 C-terminus. Mdm2 decreases the association of full-length but not C-terminally deleted p53 with a DNA target sequence in vitro and in cells. Further, using multiple approaches we demonstrate that a peptide from p53 C-terminus directly binds Mdm2 N-terminus in vitro. We also show that p300-acetylated p53 binds inefficiently to Mdm2 in vitro, and Nutlin-3 treatment induces C-terminal modification(s) of p53 in cells, explaining the low efficiency of Nutlin-3 in dissociating p53-MDM2 in vitro.

Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N-terminal coiled-coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C-terminal domain (CTD). We show that depending on pH, Gfh1-CTD exists in two alternative orientations. At pH above 7, it assumes an inactive ‘flipped' orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1-CTD switches to an active ‘Gre-like' orientation, which enables Gfh1 to bind to and inhibit RNAP.

Summary Like transcription initiation, the elongation and termination stages of transcription cycle serve as important targets for regulatory factors in prokaryotic cells. In this review, we discuss the recent progress in structural and biochemical studies of three evolutionarily conserved elongation factors, GreA, NusA and Mfd. These factors affect RNA polymerase (RNAP) processivity by modulating transcription pausing, arrest, termination or anti-termination. With structural information now available for RNAP and models of ternary elongation complexes, the interaction between these factors and RNAP can be modelled, and possible molecular mechanisms of their action can be inferred. The models suggest that these factors interact with RNAP at or near its three major, nucleic acid-binding channels: Mfd near the upstream opening of the primary (DNA-binding) channel, NusA in the vicinity of both the primary channel and the RNA exit channel, and GreA within the secondary (backtracked RNA-binding) channel, and support the view that these channels are involved in the maintenance of RNAP processivity.

RNA polymerase holoenzyme from Thermus thermophilus, consisting of six protein subunits (alpha(2), beta, beta', omega and sigma(70)) and having a total molecular mass of about 450 kDa, was purified and crystallized by the hanging-drop vapour-diffusion technique under mild near-physiological conditions. The crystals diffract beyond 3 A resolution. Careful analysis of diffraction data revealed that the crystals belong to space group P3(2), with unit-cell parameters a = b = 236.35, c = 249.04 A, and have perfect twinning along the threefold axis. A complete data set at 3 A resolution was collected and an unambiguous molecular-replacement solution was found using the structure of T. aquaticus RNA polymerase core enzyme as a search model. The refinement of structure and model building of the sigma(70) subunit is now in progress.

In bacteria, the binding of a single protein, the initiation factor sigma, to a multi-subunit RNA polymerase core enzyme results in the formation of a holoenzyme, the active form of RNA polymerase essential for transcription initiation. Here we report the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6 A resolution. In the structure, two amino-terminal domains of the sigma subunit form a V-shaped structure near the opening of the upstream DNA-binding channel of the active site cleft. The carboxy-terminal domain of sigma is near the outlet of the RNA-exit channel, about 57 A from the N-terminal domains. The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N- and C-terminal domains. The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation.

Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitt's lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, we identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader.

p53 is a tumor suppressor protein that acts as a transcription factor to regulate (either positively or negatively) a plethora of downstream target genes. Although its ability to induce protein coding genes is well documented, recent studies have implicated p53 in the regulation of non-coding RNAs, including both microRNAs (e.g. miR-34a) and long non-coding RNAs (e.g. lincRNA-p21). We have identified the non-protein coding locus PVT1 as a p53-inducible target gene. PVT1, a very large (>300 kb) locus located downstream of c-myc on chromosome 8q24, produces a wide variety of spliced non-coding RNAs as well as a cluster of six annotated microRNAs: miR-1204, miR-1205, miR-1206, miR-1207-5p, miR-1207-3p, and miR-1208. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and luciferase assays reveal that p53 binds and activates a canonical response element within the vicinity of miR-1204. Consistently, we demonstrate the p53-dependent induction of endogenous PVT1 transcripts and consequent up-regulation of mature miR-1204. Finally, we have shown that ectopic expression of miR-1204 leads to increased p53 levels and causes cell death in a partially p53-dependent manner.

The strains of Thermus thermophilus that contain the nitrate respiration conjugative element (NCE) replace their aerobic respiratory chain by an anaerobic counterpart made of the Nrc-NADH dehydrogenase and the Nar-nitrate reductase in response to nitrate and oxygen depletion. This replacement depends on DnrS and DnrT, two homologues to sensory transcription factors encoded in a bicistronic operon by the NCE. DnrS is an oxygen-sensitive protein required in vivo to activate transcription on its own dnr promoter and on that of the nar operon, but not required for the expression of the nrc operon. In contrast, DnrT is required for the transcription of these three operons and also for the repression of nqo, the operon that encodes the major respiratory NADH dehydrogenase expressed during aerobic growth. Thermophilic in vitro assays revealed that low DnrT concentrations allows the recruitment of the T. thermophilus RNA polymerase sigma(A) holoenzyme to the nrc promoter and its transcription, whereas higher DnrT concentrations are required to repress transcription on the nqo promoter. In conclusion, our data show a complex autoinducible mechanism by which DnrT functions as the transcriptional switch that allows the NCE to take the control of the respiratory metabolism of its host during adaptation to anaerobic growth.

Like transcription initiation, the elongation and termination stages of transcription cycle serve as important targets for regulatory factors in prokaryotic cells. In this review, we discuss the recent progress in structural and biochemical studies of three evolutionarily conserved elongation factors, GreA, NusA and Mfd. These factors affect RNA polymerase (RNAP) processivity by modulating transcription pausing, arrest, termination or anti-termination. With structural information now available for RNAP and models of ternary elongation complexes, the interaction between these factors and RNAP can be modelled, and possible molecular mechanisms of their action can be inferred. The models suggest that these factors interact with RNAP at or near its three major, nucleic acid-binding channels: Mfd near the upstream opening of the primary (DNA-binding) channel, NusA in the vicinity of both the primary channel and the RNA exit channel, and GreA within the secondary (backtracked RNA-binding) channel, and support the view that these channels are involved in the maintenance of RNAP processivity.

Prokaryotic transcription elongation factors GreA and GreB stimulate intrinsic nucleolytic activity of RNA polymerase (RNAP). The proposed biological role of Gre-induced RNA hydrolysis includes transcription proofreading, suppression of transcriptional pausing and arrest, and facilitation of RNAP transition from transcription initiation to transcription elongation. Using an array of biochemical and molecular genetic methods, we mapped the interaction interface between Gre and RNAP and identified the key residues in Gre responsible for induction of nucleolytic activity in RNAP. We propose a structural model in which the C-terminal globular domain of Gre binds near the opening of the RNAP secondary channel, the N-terminal coiled-coil domain (NTD) protrudes inside the RNAP channel, and the tip of the NTD is brought to the immediate vicinity of RNAP catalytic center. Two conserved acidic residues D41 and E44 located at the tip of the NTD assist RNAP by coordinating the Mg2+ ion and water molecule required for catalysis of RNA hydrolysis. If so, Gre would be the first transcription factor known to directly participate in the catalytic act of RNAP.

Publisher Summary This chapter discusses the biochemical assaying of Gre factors of Thermus Thermophilus. The biochemical studies of Escherichia coli Gre factors indicate that they are not nucleases but RNAP co-factors, which activate the same catalytic center involved in both RNA synthesis and RNA hydrolysis reactions. The biological role of factor-induced endonucleolytic reaction includes: the enhancement of transcription fidelity, by helping RNAP excise misincorporated nucleotides; suppression of transcriptional pausing and arrest by reactivation of RNAP during reversible and irreversible backtracking; and stimulation of RNAP promoter escape and transition from initiation to elongation stage of transcription by helping the catalytic center reengage with nascent RNA 3´-terminus during abortive synthesis. The chapter describes four new methods useful for biochemical and structure-functional studies of Tth Gre factors: direct chromatographic assay for competitive binding of GreA1 and GreA2 to RNAP, specific transcript cleavage (misincorporation–excision) assay for GreA1, inhibition of RNA synthesis assay for GreA2, and localized Fe2 + -induced hydroxyl radical mapping of GreA1 and GreA2 sites proximal to RNAP catalytic center.

Publisher Summary Two homologous prokaryotic proteins, GreA and GreB, L2 and a eukaryotic analog TFIIS (SII) comprise a novel class of transcription factors that are involved in endonucleolytic hydrolysis of nascent RNA in ternary complexes (TCs) of RNA polymerase (RNAP). Unlike classic RNases, SII and Gre proteins do not display any nucleolytic activity toward free RNA or RNA-DNA duplex and generally require formation of a stable TC. In the absence of factors, the intrinsic endonucleolytic activity of Escherichia coli RNAP and eukaryotic RNAP II can be stimulated by mild alkaline pH 5 and pyrophosphate, respectively. Both RNA synthesis and RNA hydrolysis reactions require at least two Mg 2+ ions with similar K m values. Moreover, the two reactions are activated and inhibited by similar groups of divalent ions, such as Mn 2+ , Fe 2+ , and Co 2+ , and Pb 2+ , Cu 2+ , and Zn 2+ , respectively. These results suggest that the same active center of RNAP is involved in both reactions. The transcript cleavage occurs when RNAP in a TC engages in “backtracking,” that is characterized by reverse translocation of RNAP together with the transcription bubble along the DNA template. Backtracking has been demonstrated both in vitro and in vivo ; however, the nature of the signals that cause it (besides destabilization of the DNA-RNA hybrid in the TC) is poorly understood.

Multiple structural, biochemical and in vivo studies have solidified the role that histone acetyltransferases (HATs) play in regulation of the p53 pathway.1 HATs are known to modulate p53 functions in many ways: from regulating its stability to promoting acetylation-dependent interactions with DNA as well as various co-factors and chromatin modifiers.1 Of these, p300, CBP, PCAF and Tip60 are the most well-studied p53 co-factors that can regulate (sometimes selectively) a number of bona fide p53 targets involved in cell cycle, apoptosis, DNA repair, metabolism and other processes.2 Of particular current interest is the time- and stress-dependent interplay between different acetyltransferases. Yet, though we now know the players, we still have only limited knowledge of their performances. A paper by Love et al. in a previous issue of Cell Cycle, has contributed new insight into the function of one such HAT.3 Using multiple p53-activating stress conditions in combination with siRNA-mediated knockdown of specific HATs in several cancer cell lines, the authors have demonstrated the dependence of p21-driven cell cycle arrest on the histone acetyl transferase activity of PCAF. They found that PCAF, but not p300 or CBP (two closely related and well characterized transcriptional co-activators) is absolutely required for maximal p21 expression in several settings. Intriguingly, their work indicates that PCAF is exclusively important for the activation of cell cycle arrest through p53- but not Rb-dependent pathways. A pictorial description of possible events leading to p21 activation is shown in Figure 1. Figure 1. Involvement of PCAF in p53-dependent activation of the p21 promoter. Upper panel: chromatin landscape and factors present at the p21 promoter in the absence of p53-activating stress signals. The p21 promoter is enriched with nucleosomes ... Key features of the paper by Love et al. are summarized as follows. First, PCAF-dependent effects on p21 transcription are apparently unrelated to its reported MDM2-directed E3 ligase activity, which otherwise would result in subsequent elevation of p53 levels.4 Second, acetylation of a previously identified PCAF site within p53, Lys320,5 is not necessary for p21 transactivation, although the acetyl-transferase activity of PCAF, per se, is indispensable. Accordingly, previously identified PCAF sites within the H3 core histone, Lys9 and Lys14, are markedly acetylated at the strong p53 “distal” binding site within the p21 promoter following stress-induced p53 activation. Since the same lysine residues are known to be acetylated by other HATs (e.g., GCN5, SRC-1 and GCN5, p300, Tip60 and SRC-1), stress- or co-factor-dependent specificity of those modifications would need to be investigated in future studies. Finally, somewhat unexpectedly, the authors did not observe any significant changes in the levels of PCAF at the distal p53 binding site within the p21 promoter before and after p53 activation. So, it is unclear what brings PCAF to the promoter of p21 gene. Detailed analysis of the nucleosome content at that region, as reported by Laptenko et al.,6 could be informative in that regard. Unlike the p300 and CBP HATs, human PCAF functions within a complex of more than 20 proteins.7 The acetyltransferase activity of the PCAF complex toward nucleosomal substrates is known to be markedly higher than of the PCAF enzyme itself. Several subunits within the complex show 100% identity to TAFs (TATA-binding protein-associated factors), while others are highly homologous but not identical to them. One such subunit, TAFII31 (a part of the TFIID complex), has already been shown to stabilize and activate p538; so this may provide some connection between p53-PCAF-dependent events at the distal p53 sites and the region of the promoter that is close to the start site. Finally, the largest subunit of the PCAF complex, TRAPP/PAF400, a member of the ATM super family and a component of the Tip60 HAT complex, may facilitate multiprotein assemblies (e.g., chromatin remodeling complexes) on targeted promoters . A good scientific study, in its attempt to answer a few specific questions, inevitably generates more questions. Among those prompted by the report by Love et al. are: what brings PCAF to the p21 promoter in the absence of high levels of p53? What other subunits/activities, if any, of the PCAF complex are vital for p21 promoter activation? What PCAF-dependent changes in chromatin occur within the p21 transcription start site following stress? Future experiments will hopefully be able to address these and other questions.

Nat. Struct. Mol. Biol. 17, 982–989 (2010); corrected online 21 September 2010 In the version of this article initially published, the error bars in Figures 1, 2, 4 and 5 were not defined, and a formatting mistake occurred in Supplementary Figure 1a. The errors have been corrected in the PDF and HTML versions of this article.

SummaryWe define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to but not overlapping the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight-bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents.