Ribosome biogenesis and translation control are essential cellular processes that are governed at numerous levels. Several tumour suppressors and proto-oncogenes have been found either to affect the formation of the mature ribosome or to regulate the activity of proteins known as translation factors. Disruption in one or more of the steps that control protein biosynthesis has been associated with alterations in the cell cycle and regulation of cell growth. Therefore, certain tumour suppressors and proto-oncogenes might regulate malignant progression by altering the protein synthesis machinery. Although many studies have correlated deregulation of protein biosynthesis with cancer, it remains to be established whether this translates directly into an increase in cancer susceptibility, and under what circumstances.

Does the ribosome translate cancer

The DNA-dependent protein kinase (DNA–PK) is anuclear serine/threonine protein kinase that is activatedupon association with DNA. Biochemical and geneticdata have revealed DNA–PK to be composed of a largecatalytic subunit, termed DNA–PKcs, and a regulatoryfactor termed Ku. In recent years, mammalian DNA–PKhas been shown to be a crucial component of both theDNA double-strand break (DSB) repair machinery andthe

/pdf/the-dna-dependent-protein-kinase-3oilvitejv.pdf

The DNA-dependent protein kinase

https://www.cell.com/cell/pdf/0092-8674(95)90360-7.pdf

Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation

https://www.bnl.gov/tcp/uploads/files/BSA01-02j.pdf

DNA-dependent protein kinase catalytic subunit: A relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product

In the budding yeast, Saccharomyces cerevisiae, genes in close proximity to telomeres are subject to transcriptional silencing through the process of telomere position effect (TPE). Here, we show that the protein Ku, previously implicated in DNA double-strand break (DSB) repair and in telomeric length maintenance, is also essential for telomeric silencing. Furthermore, using an in vivo plasmid rejoining assay, we demonstrate that SIR2, SIR3 and SIR4, three genes shown previously to function in TPE, are essential for Ku-dependent DSB repair. As is the case for Ku-deficient strains, residual repair operating in the absence of the SIR gene products ensues through an error-prone DNA repair pathway that results in terminal deletions. To identify novel components of the Ku-associated DSB repair pathway, we have tested several other candidate genes for their involvement in DNA DSB repair, telomeric maintenance and TPE. We show that TEL1, a gene required for telomeric length maintenance, is not required for either DNA DSB repair or TPE. However, RAD50, MRE11 and XRS2 function both in Ku-dependent DNA DSB repair and in telomeric length maintenance, although they have no major effects on TPE. These data provide important insights into DNA DSB repair and the linkage of this process to telomere length homeostasis and transcriptional silencing.

/pdf/components-of-the-ku-dependent-non-homologous-end-joining-4xr5ecx70i.pdf

Components of the Ku‐dependent non‐homologous end‐joining pathway are involved in telomeric length maintenance and telomeric silencing

DNA-dependent protein kinase (DNA-PK) comprises a catalytic subunit of -350 kD (p350) and a DNA-binding component termed Ku. Although DNA-PK can phosphorylate many transcription factors, no function for this enzyme in transcription has been reported thus far. Here, we show that DNA-PK strongly represses transcription by RNA polymerase I (Pol I). Transcriptional repression by DNA-PK requires ATP hydrolysis, and DNA-PK must be colocalized on the same DNA molecule as the Pol I transcription machinery. Consistent with DNA-PK requiring DNA ends for activity, transcriptional inhibition only occurs effectively on linearized templates. Mechanistic studies including single-round transcriptions, abortive initiation assays, and factor-independent transcription on a tailed template demonstrate that DNA-PK inhibits initiation (i.e., the formation of the first phosphodiester bonds) but does not affect transcription elongation. Repression of transcription involves phosphorylation of the transcription initiation complex, and rescue experiments reveal that the inactivated factor remains bound to the promoter and thus prevents initiation complex formation. We discuss the possible relevance of these findings in regard to the control of rRNA synthesis in vivo.

/pdf/dna-dependent-protein-kinase-a-potent-inhibitor-of-47wm5rj6qm.pdf

DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I.

The nucleolar factor UBF is phosphorylated by casein kinase II (CKII) at serine residues within the C-terminal acidic domain which is required for transcription activation. To investigate the biological significance of UBF modification, we have compared the trans-activating properties of cellular UBF and recombinant UBF expressed in Escherichia coli. Using a variety of assays we demonstrate that unphosphorylated UBF is transcriptionally inactive and has to be phosphorylated at multiple sites to stimulate transcription. Examination of cDNA mutants in which the serine residues within the C-terminal domain were altered by site-directed mutagenesis demonstrates that CKII-mediated phosphorylations of UBF contribute to, but are not sufficient for, transcriptional activation. Besides CKII, other cellular protein kinases phosphorylate UBF at distinct sites in a growth-dependent manner. The marked differences in the tryptic peptide maps of UBF from growing and serum-starved cells suggest that alterations in the degree of UBF phosphorylation may modulate rRNA synthetic activity in response to extracellular signals.

Activation of mammalian ribosomal gene transcription requires phosphorylation of the nucleolar transcription requires phosphorylation of the nucleolar trasnscription factor UBF

THE 18-base-pair sequence element AGGTCGACCAGTACTCCG (the Sal box) signals termination of mouse ribosomal gene transcription1. This sequence is recognized by a sequence-specific DNA-binding protein, TTF I, which mediates the termination of transcription by RNA polymerase I (poi I) 2,3. Subsequently, the ends of the primary transcripts are trimmed by 10 nucleotides in a sequence-dependent 3′-terminal processing reaction4. We have now investigated whether TTF I bound to its target sequence will block elongation by any RNA polymerase by steric hindrance, or whether it is specific for elongation by pol I. The results demon-strate that TTF I directs transcription termination with RNA poly-merase I from species as divergent as mouse and yeast, but fails to affect elongation by heterologous polymerases (eukaryotic RNA polymerases II and III, Escherichia coli or bacteriophage T3 RNA polymerase). By contrast, purified lac represser bound to its operator sequence stops elongation by both RNA polymerase I and II.

Specific interaction of the murine transcription termination factor TTF I with class-I RNA polymerases.

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.

The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription

We show that the repetitive 140-base-pair (bp) elements present in the spacer of mouse rRNA genes function as enhancers for RNA polymerase I. Attachment of these elements to the rDNA promoter stimulates rRNA synthesis both in vivo and in vitro. The cis-activating effect of the spacer repeats is orientation-independent and increases with increasing numbers of the 140-bp elements. Competition experiments demonstrate that the spacer repeats bind one or more of the transcription factors interaction with the rDNA promoter. Both the 140-bp elements and the core promoter act cooperatively and thus are functionally linked. The 60/81-bp enhancer repeats from Xenopus laevis rDNA compete for a murine transcription factor(s) and stimulate transcription often fused to the mouse rDNA promoter. The results indicate that despite the marked species specificity of rDNA transcription initiation, common factors may interact with both the rDNA promoter and the enhancer.

Anne Kuhn

Papers

DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I.

Activation of mammalian ribosomal gene transcription requires phosphorylation of the nucleolar transcription requires phosphorylation of the nucleolar trasnscription factor UBF

Specific interaction of the murine transcription termination factor TTF I with class-I RNA polymerases.

The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription

A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system.