The eukaryotic upstream binding factor (UBF), recognizes the ribosomal RNA gene promoter and activates transcription mediated by RNA polymerase I through cooperative interactions with the species-specific factor, SL1. Isolation of complementary DNA clones and sequence analysis reveals similarities between DNA binding domains of human UBF (hUBF) and high mobility group (HMG) protein 1. Expression, cellular localization and in vitro transcription studies establish that cloned hUBF encodes a nucleolar factor that binds specifically to the upstream control element and core of the rRNA gene promoter to activate transcription in a binding site-dependent manner.

c-Myc coordinates cell growth and division through a transcriptional programme that involves both RNA polymerase (Pol) II- and Pol III-transcribed genes. Here, we demonstrate that human c-Myc also directly enhances Pol I transcription of ribosomal RNA (rRNA) genes. rRNA synthesis and accumulation occurs rapidly following activation of a conditional MYC-ER allele (coding for a Myc-oestrogen-receptor fusion protein), is resistant to inhibition of Pol II transcription and is markedly reduced by c-MYC RNA interference. Furthermore, by using combined immunofluorescence and rRNA-FISH, we have detected endogenous c-Myc in nucleoli at sites of active ribosomal DNA (rDNA) transcription. Our data also show that c-Myc binds to specific consensus elements located in human rDNA and associates with the Pol I-specific factor SL1. The presence of c-Myc at specific sites on rDNA coincides with the recruitment of SL1 to the rDNA promoter and with increased histone acetylation. We propose that stimulation of rRNA synthesis by c-Myc is a key pathway driving cell growth and tumorigenesis.

We have previously shown that the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs) are required for regulated transcriptional initiation by RNA polymerase II. Here we report the biochemical properties of the RNA polymerase I promoter selectivity factor, SL1, and its relationship to TBP. Column chromatography and glycerol gradient sedimentation indicate that a subpopulation of TBP copurifies with SL1 activity. Antibodies directed against TBP efficiently deplete SL1 transcriptional activity, which can be restored with the SL1 fraction but not purified TBP. Thus, TBP is necessary but not sufficient to complement SL1 activity. Analysis of purified SL1 reveals a complex containing TBP and three distinct TAFs. Purified TAFs reconstituted with recombinant TBP complement SL1 activity, and this demonstrates that TBP plus novel associated factors are integral components of SL1. These findings suggest that TBP may be a universal transcription factor and that the TBP-TAF arrangement provides a unifying mechanism for promoter recognition in animal cells.

The human ribosomal RNA promoter contains two distinct control elements (UCE and core) both of which are recognized by the sequence-specific DNA binding protein UBF1, which has now been purified to apparent homogeneity. The purified factor activates RNA polymerase I (RNA pol I) transcription through direct interactions with either control element. A second RNA pol I transcription factor, designated SL1, participates in the promoter recognition process and is required to reconstitute transcription in vitro. Although SL1 alone has no sequence-specific DNA binding activity, deoxyribonuclease I footprinting experiments reveal that a cooperative interaction between UBF1 and SL1 leads to the formation of a new protein-DNA complex at the UCE and core elements. In vitro transcription experiments indicate that formation of the UBF1-SL1 complex is vital for transcriptional activation by UBF1. Thus, protein-protein interactions between UBF1 and SL1 are required for targeting of SL1 to cis-control sequences of the promoter.

The RNA genome of the human immunodeficiency virus type-1 (HIV-1) contains a approximately 120 nucleotide Psi-packaging signal that is recognized by the nucleocapsid (NC) domain of the Gag polyprotein during virus assembly. The Psi-site contains four stem-loops (SL1-SL4) that possess overlapping and possibly redundant functions. The present studies demonstrate that the 19 residue SL2 stem-loop binds NC with affinity (K(d)=110(+/-50) nM) similar to that observed for NC binding to SL3 (K(d)=170(+/-65) nM) and tighter than expected on the basis of earlier work, suggesting that NC-SL2 interactions probably play a direct role in the specific recognition and packaging of the full-length, unspliced genome. The structure of the NC-SL2 complex was determined by heteronuclear NMR methods using (15)N,(13)C-isotopically labeled NC protein and SL2 RNA. The N and C-terminal "zinc knuckles" (Cys-X(2)-Cys-X(4)-His-X(4)-Cys; X=variable amino acid) of HIV-1 NC bind to exposed guanosine bases G9 and G11, respectively, of the G8-G9-U10-G11 tetraloop, and residues Lys3-Lys11 of the N-terminal tail forms a 3(10) helix that packs against the proximal zinc knuckle and interacts with the RNA stem. These structural features are similar to those observed previously in the NMR structure of NC bound to SL3. Other features of the complex are substantially different. In particular, the N-terminal zinc knuckle interacts with an A-U-A base triple platform in the minor groove of the SL2 RNA stem, but binds to the major groove of SL3. In addition, the relative orientations of the N and C-terminal zinc knuckles differ in the NC-SL2 and NC-SL3 complexes, and the side-chain of Phe6 makes minor groove hydrophobic contacts with G11 in the NC-SL2 complex but does not interact with RNA in the NC-SL3 complex. Finally, the N-terminal helix of NC interacts with the phosphodiester backbone of the SL2 RNA stem mainly via electrostatic interactions, but does not bind in the major groove or make specific H-bonding contacts as observed in the NC-SL3 structure. These findings demonstrate that NC binds in an adaptive manner to SL2 and SL3 via different subsets of inter and intra-molecular interactions, and support a genome recognition/packaging mechanism that involves interactions of two or more NC domains of assembling HIV-1 Gag molecules with multiple Psi-site stem-loop packaging elements during the early stages of retrovirus assembly.

Retrovirus particles each contain two copies of the viral genome in the form of a noncovalently linked RNA dimer. Earlier studies have mapped a cis-acting region near the 5' end of the human immunodeficiency virus type 1 (HIV-1) genome, termed the psi locus, which appears essential for initiation of genomic dimerization, as well as for interactions with the HIV-1 Gag protein that are thought to target the RNA into nascent virions. This HIV-1 psi locus is proposed to be organized in four independent RNA stem-loops; at least three (SL1, SL3, and SL4) contain binding sites for Gag, and one of these (SL1) is implicated in dimer initiation through a kissing-loop mechanism. In this study, we have created HIV-1 proviruses containing psi mutations that affect in vitro Gag binding, RNA dimerization, or both, and we have characterized the effects of these mutations on viral assembly and infectivity by using a single-step infectious assay. We find that various mutations which eliminate the Gag binding sites in SL1 or SL3 produce marked defects in genomic RNA packaging and viral infectivity. In each case, the reduced genomic content of the mutant virions is associated with an increased content of spliced viral transcripts, suggesting that both SL1 and SL3 contribute to the discrimination between spliced and unspliced RNAs. The structures, but not the specific sequences, of the SL1 and SL3 stems appear critical for RNA packaging. Disruption of the stem or deletion of SL1 also results in abnormal genomic dimerization, as assessed by nondenaturing gel electrophoresis of virion-derived RNA. Virions carrying less extensive mutations in the SL1 loop that are known to prevent in vitro dimerization have impaired infectivity despite normal virion RNA content. This suggests that RNA dimerization is not a prerequisite for genomic packaging but instead serves an independent function in the retroviral infectious cycle.

Regulation of ribosomal RNA genes is a fundamental process that supports the growth of cells and is tightly coupled with cell differentiation. Although rRNA transcriptional control by RNA polymerase I (Pol I) and associated factors is well studied, the lineage-specific mechanisms governing rRNA expression remain elusive. Runt-related transcription factors Runx1, Runx2 and Runx3 establish and maintain cell identity, and convey phenotypic information through successive cell divisions for regulatory events that determine cell cycle progression or exit in progeny cells. Here we establish that mammalian Runx2 not only controls lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II, but also acts as a repressor of RNA Pol I mediated rRNA synthesis. Within the condensed mitotic chromosomes we find that Runx2 is retained in large discrete foci at nucleolar organizing regions where rRNA genes reside. These Runx2 chromosomal foci are associated with open chromatin, co-localize with the RNA Pol I transcription factor UBF1, and undergo transition into nucleoli at sites of rRNA synthesis during interphase. Ribosomal RNA transcription and protein synthesis are enhanced by Runx2 deficiency that results from gene ablation or RNA interference, whereas induction of Runx2 specifically and directly represses rDNA promoter activity. Runx2 forms complexes containing the RNA Pol I transcription factors UBF1 and SL1, co-occupies the rRNA gene promoter with these factors in vivo, and affects local chromatin histone modifications at rDNA regulatory regions. Thus Runx2 is a critical mechanistic link between cell fate, proliferation and growth control. Our results suggest that lineage-specific control of ribosomal biogenesis may be a fundamental function of transcription factors that govern cell fate.

The mRNAs of six C. elegans genes are known to be trans-spliced to SL2. We report here that a similarly oriented gene is located 100-300 bp upstream of each. We present evidence that the genes in these clusters are cotranscribed and downstream mRNAs are formed by cleavage at the polyadenylation site and trans-splicing. From one three-gene cluster we isolated cDNA clones representing both polycistronic RNAs and mRNAs polyadenylated at the free 3' end created by trans-splicing, suggesting that polycistronic RNAs can be processed by trans-splicing. Several experiments indicate that SL2 trans-splicing is a consequence of a gene's downstream location in an operon. In particular, when an SL1-accepting gene was moved to a downstream location, its mRNA was trans-spliced largely to SL2. The possible regulatory significance of cotranscription of C. elegans genes is discussed.

The tumor suppressor protein p53 is frequently inactivated in tumors. It functions as a transcriptional activator as well as a repressor for a number of viral and cellular promoters transcribed by RNA polymerase II (Pol II) and by RNA Pol III. Moreover, it appears that p53 also suppresses RNA Pol I transcription. In this study, we examined the molecular mechanism of Pol I transcriptional inhibition by p53. We show that wild-type, but not mutant, p53 can repress Pol I transcription from a human rRNA gene promoter in cotransfection assays. Furthermore, we show that recombinant p53 inhibits rRNA transcription in a cell-free transcription system. In agreement with these results, p53-null epithelial cells display an increased Pol I transcriptional activity compared to that of epithelial cells that express p53. However, both cell lines display comparable Pol I factor protein levels. Our biochemical analysis shows that p53 prevents the interaction between SL1 and UBF. Protein-protein interaction assays indicate that p53 binds to SL1, and this interaction is mostly mediated by direct contacts with TATA-binding protein and TAF(I)110. Moreover, template commitment assays show that while the formation of a UBF-SL1 complex can partially relieve the inhibition of transcription, only the assembly of a UBF-SL1-Pol I initiation complex on the rDNA promoter confers substantial protection against p53 inhibition. In summary, our results suggest that p53 represses RNA Pol I transcription by directly interfering with the assembly of a productive transcriptional machinery on the rRNA promoter.

Although eukaryotic genes are usually transcribed individually, at least a few Caenorhabditis elegans genes appear to be transcribed polycistronically in clusters resembling bacterial operons. The spliced leader SL2 (ref. 2) is specific for trans-splicing to downstream genes in these operons. In addition, many C. elegans pre-mRNAs are trans-spliced to SL1 (ref. 3) near the 5' ends of pre-mRNAs. Because operons have not previously been found in higher eukaryotes, we have investigated how widespread they are in the C. elegans genome. We identified gene clusters using the extensive data generated by the genome project and tested seven for trans-splicing specificity. All were found to fit expectations for polycistronic transcription. In addition, we surveyed reported C. elegans genes for trans-splicing specificity. Both methods indicate that the pre-mRNAs of about 70% of C. elegans genes are trans-spliced and as many as a quarter are transcribed in operons.

Sequences from the 5' end of type 1 human immunodeficiency virus RNA dimerize spontaneously in vitro in a reaction thought to mimic the initial step of genomic dimerization in vivo. Dimer initiation has been proposed to occur through a "kissing-loop" interaction involving a specific RNA stem-loop element designated SL1: the RNA strands first interact by base pairing through a six-base GC-rich palindrome in the loop of SL1, whose stems then isomerize to form a longer interstrand duplex. We now report a mutational analysis aimed at defining the features of SL1 RNA sequence and secondary structure required for in vitro dimer formation. Our results confirm that mutations which destroy complementarity in the SL1 loop abolish homodimer formation, but that certain complementary loop mutants can heterodimerize. However, complementarity was not sufficient to ensure dimerization, even between GC-rich loops, implying that specific loop sequences may be needed to maintain a conformation that is competent for initial dimer contact; the central GC pair of the loop palindrome appeared critical in this regard, as did two or three A residues which normally flank the palindrome. Neither the four-base bulge normally found in the SL1 stem nor the specific sequence of the stem itself was essential for the interaction; however, the stem structure was required, because interstrand complementarity alone did not support dimer formation. Electron microscopic analysis indicated that the RNA dimers formed in vitro morphologically resembled those isolated previously from retroviral particles. These results fully support the kissing-loop model and may provide a framework for systematically manipulating genomic dimerization in type 1 human immunodeficiency virus virions.

The human rRNA promoter contains two distinct cis-control sequences, the core and upstream control element (UCE), that serve as the target for binding cellular trans-activating proteins involved in transcription initiation by RNA polymerase I. One of these factors, SL1, has been extensively purified and shown to be a species-specific factor required to reconstitute in vitro RNA synthesis. DNAase footprinting revealed that although SL1 alone does not bind specifically to rRNA promoter sequences, a second factor, UBF1, recruits SL1 to the template and directs binding to an extended region encompassing sequences in the UCE. Analysis of mutant and human-mouse hybrid promoters indicate that protein-DNA interactions at the UCE modulate the efficiency of rRNA synthesis. Transcription from the human rRNA promoter appears to require an unusual set of protein-DNA transactions in which recognition and binding to an upstream cis-control element is carried out by one factor (UBF1), whereas activation requires an additional factor, SL1, acting in conjunction with UBF1 to trigger transcription.

Cells respond to a variety of extracellular and intracellular forms of stress by down-regulating rRNA synthesis. We have investigated the mechanism underlying stress-dependent inhibition of RNA polymerase I (Pol I) transcription and show that the Pol I-specific transcription factor TIF-IA is inactivated upon stress. Inactivation is due to phosphorylation of TIF-IA by c-Jun N-terminal kinase (JNK) at a single threonine residue (Thr 200). Phosphorylation at Thr 200 impairs the interaction of TIF-IA with Pol I and the TBP-containing factor TIF-IB/SL1, thereby abrogating initiation complex formation. Moreover, TIF-IA is translocated from the nucleolus into the nucleoplasm. Substitution of Thr 200 by valine as well as knock-out of Jnk2 prevent inactivation and translocation of TIF-IA, leading to stress-resistance of Pol I transcription. Our data identify TIF-IA as a downstream target of the JNK pathway and suggest a critical role of JNK2 to protect rRNA synthesis against the harmful consequences of cellular stress.

A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.

The TATA box-binding protein TBP directs transcription by all three eukaryotic RNA polymerases. In mammalian cells, TBP is found in at least three different complexes: SL1, D-TFIID, and B-TFIID. While SL1 and D-TFIID are involved in RNA polymerase I and II transcription, respectively, no unique function has been assigned to the B-TFIID complex. Here we show that the TFIIIB fraction required for RNA polymerase III transcription contains two separable components, one of which is a TBP-containing complex that may correspond to B-TFIID. For transcription of TATA-less RNA polymerase III genes such as the VAI, 5S, and 7SL genes, this complex cannot be replaced by either TBP alone or the D-TFIID complex. In contrast, TBP alone is active for basal transcription from the TATA-containing U6 promoter. This indicates different requirements for recruiting TBP to TATA-less and TATA-containing RNA polymerase III promoters.

In the human small nuclear RNA (snRNA) promoters, the presence of a TATA box recognized by the TATA box-binding protein (TBP) determines the selection of RNA polymerase III over RNA polymerase II. The RNA polymerase II snRNA promoters are, therefore, good candidates for TBP-independent promoters. We show here, however, that TBP activates transcription from RNA polymerase II snRNA promoters through a non-TATA box element, the snRNA proximal sequence element (PSE), as part of a new snRNA-activating protein complex (SNAPc). In contrast to the previously identified TBP-containing complexes SL1, TFIID, and TFIIIB, which appear dedicated to transcription by a single RNA polymerase, SNAPc is also essential for RNA polymerase III transcription from the U6 snRNA promoter. The U6 initiation complex appears to contain two forms of TBP, one bound to the TATA box and one bound to the PSE as a part of SNAPc, suggesting that multiple TBP molecules can have different functions within a single promoter.

A crucial step in transcription is the recruitment of RNA polymerase to promoters. In the transcription of human rRNA genes by RNA Polymerase I (Pol I), transcription factor SL1 has a role as the essential core promoter binding factor. Little is known about the mechanism by which Pol I is recruited. We provide evidence for an essential role for hRRN3, the human homologue of a yeast Pol I transcription factor, in this process. We find that whereas the bulk of human Pol I complexes (I alpha) are transcriptionally inactive, hRRN3 defines a distinct subpopulation of Pol I complexes (I beta) that supports specific initiation of transcription. Human RRN3 interacts directly with TAF(I)110 and TAF(I)63 of promoter-selectivity factor SL1. Blocking this connection prevents recruitment of Pol I beta to the rDNA promoter. Furthermore, hRRN3 can be found in transcriptionally autonomous Pol I holoenzyme complexes. We conclude that hRRN3 functions to recruit initiation-competent Pol I to rRNA gene promoters. The essential role for hRRN3 in linking Pol I to SL1 suggests a mechanism for growth control of Pol I transcription.

AMP-activated protein kinase (AMPK) senses changes in the intracellular AMP/ATP ratio, switching off energy-consuming processes and switching on catabolic pathways in response to energy depletion. Here, we show that AMPK down-regulates rRNA synthesis under glucose restriction by phosphorylating the RNA polymerase I (Pol I)-associated transcription factor TIF-IA at a single serine residue (Ser-635). Phosphorylation by AMPK impairs the interaction of TIF-IA with the TBP-containing promoter selectivity factor SL1, thereby precluding the assembly of functional transcription initiation complexes. Mutation of Ser-635 compromises down-regulation of Pol I transcription in response to low energy supply, supporting that activation of AMPK adapts rRNA synthesis to nutrient availability and the cellular energy status.

Three familial variants of the presynaptic protein alpha-synuclein (alphaS), A30P, E46K, and A53T, correlate with rare inherited Parkinson's disease (PD), while wild-type alphaS is implicated in sporadic PD. The classic manifestation of both familiar and sporadic PD is the formation of fibrillar structures of alphaS which accumulate as the main component in intraneuronal Lewy bodies. At presynaptic termini, the partitioning of alphaS between disordered cytosolic and membrane-bound states likely mediates its proposed role in regulation of reserve pools of synaptic vesicles. Previously, we reported on multiple distinct phospholipid binding modes of alphaS with slow binding kinetics. Here, we report the phospholipid binding properties of the disease variants, viewed by solution NMR in a residue-specific manner. Our results agree qualitatively with previous biophysical studies citing overall decreased lipid affinity for the A30P mutation, comparable affinity for A53T, and an increased level of binding of E46K, relative to wild-type alphaS. Additionally, our NMR results describe the distribution of lipid-bound states for alphaS: the population of the SL1 binding mode (residues 3-25 bound as a helix) is augmented by each of the disease variants, relative to wild-type alphaS. We propose that the SL1 binding mode, which anchors the N-terminus of alphaS in the lipoprotein complex while the hydrophobic NAC region remains dynamically disordered, is prone to intermolecular interactions which progress toward disease-associated oligomers and fibrils. The elevation of the SL1 binding mode, unchecked by a proportionate population of binding modes incorporating the full N-terminal domain, may well account for the increased toxicity of the A30P, E46K, and A53T disease variants of alphaS.

How can trans-activators with the same DNA binding specificity direct different transcriptional programs? The rRNA transcriptional apparatus offers a useful model system to address this question and to dissect the mechanisms that generate alternative transcription complexes. Here, we compare the mouse and human transcription factors that govern species-specific RNA polymerase I promoter recognition. We find that both human and mouse rRNA transcription is mediated by a specific multiprotein complex. One component of this complex is the DNA-binding transcription factor, UBF. Paradoxically, human and mouse UBF display identical DNA binding specificities even though transcription of rRNA is species specific. Promoter selectivity is conferred by a second essential factor, SL1, which, for humans, does not bind DNA independently but, instead, cooperates with UBF in the formation of high-affinity DNA-binding complexes. In contrast, mouse SL1 can selectively interact with DNA in the absence of UBF. Reconstituted transcription experiments establish that UBF and RNA polymerase I from the two species are functionally interchangeable, whereas mouse and human SL1 exhibit distinct DNA binding and transcription activities. Together, these results suggest a critical role for a specific multiprotein assembly in RNA polymerase I promoter recognition and reveal distinct mechanisms through which such complexes can generate functional diversity.

Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 5' ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of ∼70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not trans-spliced to both, indicating that SL1 and SL2 trans-splicing use different underlying mechanisms. SL2 trans-splicing occurs in order to separate the products of genes in operons genome wide. Shorter intercistronic distance is associated with greater use of SL2. Finally, increased use of SL1 trans-splicing to downstream operon genes can indicate the presence of an extra promoter in the intercistronic region, creating what has been termed a "hybrid" operon. Within hybrid operons the presence of the two promoters results in the use of the two SL classes: Transcription that originates at the promoter upstream of another gene creates a polycistronic pre-mRNA that receives SL2, whereas transcription that originates at the internal promoter creates transcripts that receive SL1. Overall, our data demonstrate that >17% of all C. elegans genes are in operons.

Caenorhabditis elegans engages in three distinct versions of nuclear pre-mRNA splicing: cis-splicing of introns and two kinds of trans-splicing that result in the addition of two different spliced leaders onto mRNAs. One leader (SL1) is used near the 5' ends of pre-mRNAs while the other (SL2) is used at internal trans-splice sites of polycistronic pre-mRNAs. Here, I consider how these three types of splicing event are faithfully carried out.

RNA polymerases I, II, and III require the TATA-binding protein (TBP) to initiate promoter-specific transcription. We have separated HeLa TBP into four phosphocellulose fractions that elicit polymerase specificity in supplying TBP activity to TBP-depleted pol II and pol III transcription reactions. Polymerase specificity might arise in part through distinct TBP-associated factors (TAFs), which have recently been identified in pol I and II transcription. However, the requirement for pol III TAFs has not been established. Here we show that classical pol III transcription involves a minimum of two novel TAFs: TAF-172 and TAF-L. Not only does TAF-172 activate pol III transcription, but it also inhibits the binding of TBP to the TATA box, thereby repressing pol II transcription. The TBP-TAF-172-TAF-L complex can replace TFIIIB both in transcription reactions reconstituted with TFIIIC and in template commitment assays. Thus SL1, TFIID, and TFIIIB might be functionally similar TBP-TAF complexes that direct pol I, II, and III transcription, respectively.

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.