. Ribosome biogenesis centres both physically and functionally on the activity of the ribosomal RNA (rRNA) genes. Ribosome assembly occurs co-transcriptionally on these genes, requires the coordinated expression and assembly of many hundreds of proteins and is finely tuned to cell and organism growth. This review presents contemporary understanding of the mode and the means of rRNA gene transcription and how growth factors, oncogenes and tumour suppressors regulate this transcription. It is argued that transcription elongation is a key mechanism regulating rRNA gene transcription. This unorthodox view provides a logical framework to explain the co-transcriptional phase of ribosome biogenesis.

Synthesis of the 45S rRNA by RNA polymerase I limits cell growth. Knowledge of the mechanism of its regulation is therefore key to understanding growth control. rRNA transcription is believed to be regulated solely at initiation/promoter release. However, we found that stimulation of endogenous 45S rRNA synthesis by epidermal growth factor (EGF) and serum failed to induce an increase in RNA polymerase I engagement on the rRNA genes, despite robust enhancement of 45S rRNA synthesis. Further, endogenous transcription elongation rates were measured and found to be directly proportional to 45S rRNA synthesis. Thus, elongation is a rate-limiting step for rRNA synthesis in vivo. ERK phosphorylation of the HMG boxes of UBF, an RNA polymerase I factor essential for transcription enhancement, was shown to directly regulate elongation by inducing the remodeling of ribosomal gene chromatin. The data suggest a mechanism forcoordinating the cotranscriptional assembly of preribosomal particles.

Transcription of the ribosomal RNA genes of mammals by RNA polymerase I is rapidly activated by epidermal growth factor via the MAP-kinase (ERK) signaling cascade. This activation is mediated by direct phosphorylation of the HMG box DNA binding domains of the architectural transcription factor UBF. Mutation of the ERK sites of UBF inhibits its normal function and blocks growth factor activation of ribosomal transcription. UBF has little or no DNA sequence selectivity and binds throughout the ribosomal genes, defining a specialized chromatin. Indeed, the HMG boxes of UBF induce looping of the ribosomal DNA to create the enhancesome, a structure somewhat reminiscent of the nucleosome. Here, we show that both ERK phosphorylation and mutations that simulate this phosphorylation decrease the affinity of the individual HMG boxes of UBF for linear ribosomal DNA but have little or no effect on the capacity of these HMG boxes to bind to pre-bent DNA and do not affect the overall binding constant of UBF for the DNA. Electron spectroscopic imaging showed that ERK site UBF mutants do not induce the characteristic DNA looping of the enhancesome and associate with no more than half of the enhancesomal DNA. The data demonstrate that ERK phosphorylation of UBF prevents DNA bending by its first two HMG boxes, leading to a cooperative unfolding, of the enhancesome.

Ribosome assembly occurs co-transcriptionally on the rRNA genes. This process requires the co-ordinated expression and assembly of many hundreds of proteins and is finely tuned to cell and organism growth. Co-ordinate regulation of the rRNA genes and the ribosomal protein genes is therefore essential for high-fidelity ribosome assembly. Recent work shows that rRNA gene transcription is regulated at the level of elongation via the mitogen-activated protein kinase pathway. We argue that this may provide an explanation for the high fidelity of ribosome assembly.

The ribosomal RNA genes encode the enzymatic scaffold of the ribosome and thereby perform perhaps the most basic of all housekeeping functions. However, recent data suggests that they might also control important aspects of cell behavior.

The so-called upstream binding factor (UBF) is required for the initial step in formation of an RNA polymerase I initiation complex. This function of UBF correlates with its ability to induce the ribosomal enhancesome, a structure which resembles in its mass and DNA content the nucleosome of chromatin. DNA looping in the enhancesome is probably the result of six in-phase bends induced by the HMG boxes of a UBF dimer. Here we show that insertion/deletion mutations in the basic peptide linker lying between the N-terminal dimerisation domain and the first HMG box of Xenopus UBF prevent the DNA looping characteristic of the enhancesome. Using these mutants we demonstrate that (i) the enhancesome structure does not depend on tethering of the entering and exiting DNA duplexes, (ii) UBF monomers induce hemi-enhancesomes, bending the DNA by 175 +/- 24 degrees and (iii) two hemi-enhancesomes are precisely phased by UBF dimerisation. We use this and previous data to refine the existing enhancesome model and show that HMG boxes 1 and 2 of UBF lie head-to-head along the DNA.

RNA polymerase I (PolI) transcription is activated by the HMG box architectural factor UBF, which loops approximately 140 bp of DNA into the enhancesome, necessitating major chromatin remodeling. Here we show that the acetyltransferase CBP is recruited to and acetylates UBF both in vitro and in vivo. CBP activates PolI transcription in vivo through its acetyltransferase domain and acetylation of UBF facilitates transcription derepression and activation in vitro. CBP activation and Rb suppression of ribosomal transcription by recruitment to UBF are mutually exclusive, regulating in vivo PolI transcription through an acetylation-deacetylation "flip-flop." Thus, PolI transcription is regulated by protein acetylation, and the competitive recruitment of CBP and Rb.

A novel RNA polymerase I (RPI) driven reporter gene has been used to investigate the in vivo role of the architectural ribosomal transcription factor UBF in gene activation and species specificity. It is shown that the level of UBF overexpression in NIH3T3 cells leads to a proportionate increase in the activities of both reporter and endogenous ribosomal genes. Further, co-expression of UBF antisense RNA suppresses reporter gene expression. Thus, UBF is limiting for ribosomal transcription in vivo and represents a potential endogenous ribosomal gene regulator. In contrast to some in vitro studies, in vivo, the mammalian and Xenopus forms of UBF1 show an equal ability to activate a mouse RPI promoter. This activity is severely impaired in mutants compromised for either dimerization or DNA binding. Similarly, the natural UBF2 splice variant shows a severely impaired capacity to activate RPI transcription. The data strongly suggest that UBF predominantly regulates ribosomal transcription by binding to and activating the ribosomal genes, but does not eliminate a possible secondary role in titrating ribosomal gene repressors such as Rb. Consistent with the DNA folding ability and cellular abundance of the UBF, we suggest that the protein may regulate a structural transition between the potentially active and active chromatin states.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.

The formation of a near complete loop of DNA is a striking property of the architectural HMG-box factor xUBF. Here we show that DNA looping only requires a dimer of Nbox13, a C-terminal truncation mutant of xUBF containing just HMG-boxes 1-3. This segment of xUBF corresponds to that minimally required for activation of polymerase I transcription and is sufficient to generate the major characteristics of the footprint given by intact xUBF. Stepwise reduction in the number of HMG-boxes to less than three significantly diminishes DNA bending and provides an estimate of bend angle for each HMG-box. Together the data indicate that a 350 +/- 16 degree loop in 142 +/- 30 bp of DNA can be induced by binding of the six HMG-boxes in an Nbox13 dimer and that DNA looping is probably achieved by six in-phase bends. The positioning of each HMG-box on the DNA does not predominantly involve DNA sequence recognition and is thus an intrinsic property of xUBF.

This chapter discusses the mechanisms of eukaryotic ribosomal transcription and its regulation. Ribosomal transcription has been studied in an exceptionally wide range of organisms. In eukaryotes, ribosomal gene transcription uses a dedicated set of transcription factors and a specialized ribonucleic acid (RNA) polymerase, the deoxyribonucleic acid (DNA)-dependent RNA polymerase I (RPOI). The resultant precursor-rRNA (pre-rRNA) is neither capped nor polyadenylated and is produced in the nucleolus; a large nuclear structure visible in the light microscope. Moreover, the promotion of ribosomal transcription is generally directed, by a 100- to 150-bp DNA sequence that lies across and, predominantly, upstream of the transcription-initiation site. Promotion requires an activated form of RPOI, the DNA-binding factor upstream transcription factor (UBF), and the TATA binding protein (TBP)I-complex, containing three TATA-box binding protein associated protein (TAF)Is. Upstream binding factor (UBF) binds to both an upstream and an initiation-site promoter element, probably coiling the promoter into a 180-bp loop. This allows the interaction of the TBP,-complex, and subsequent polymerase recruitment. The activity of the promoter is enhanced by a promoter-proximal terminator, by enhancer repeats in the proximal IGS, and by the presence in the IGS of duplicate spacer promoters. Further upstream, other sequences may also modulate transcription.

The nucleolar transcription factor UBF consists of two proteins, UBF1 and UBF2, which originate by alternative splicing. Here we show that deletion of 37 amino acids within the second of five HMG box motifs in UBF2 is important for the dual role of UBF as transcriptional activator and antirepressor. UBF1 is a potent antirepressor and transcriptional activator, whereas the ability of UBF2 to counteract histone H1‐mediated repression and to stimulate ribosomal gene transcription both in vivo and in vitro is at least one order of magnitude lower. The difference in transcriptional activity between UBF1 and UBF2 is due to their different binding to the ribosomal gene promoter and enhancer. Apparently, the presence of an intact HMG box2 modulates the sequence‐specific binding of UBF to rDNA control elements. However, the interaction of UBF with rDNA does not entirely depend on sequence recognition. Both UBF isoforms bind efficiently to four‐way junction DNA, indicating that they recognize defined DNA structures rather than specific sequences. The results demonstrate that the HMG boxes are functionally diverse and that HMG box2 plays an important role in specific binding of UBF to rDNA.

Previously we have shown that the RNA polymerase I (Pol I)-specific transcription factor UBF stimulates transcription by both facilitating transcription complex formation and by relieving repression exerted by a negative-acting factor which competes for binding of the murine factor TIF-IB to the ribosomal gene promoter (1). We have purified and functionally characterized this repressor protein from Ehrlich ascites cells. The final preparation contained two polypeptides with molecular masses of 75 and 90 kDa, respectively. Both polypeptides interact with the rDNA promoter as revealed by UV-crosslinking experiments. The specificity of binding to the ribosomal gene promoter was demonstrated in an electrophoretic mobility shift assay and by DNase footprinting. The biochemical properties of this negative-acting factor closely resemble those of the Ku antigen, a human nuclear DNA-binding heterodimer which is the target of autoantibodies in several autoimmune diseases. Anti-Ku antibodies precipitate the repressor activity and overcome transcription inhibition. The data demonstrate that regulation of Pol I gene transcription may involve an antirepression mechanism as already documented for Pol II genes and suggest that Ku protein may be causally involved in repressor-mediated down regulation of rRNA synthesis.