scholarly journals Transcriptional Activity and Chromatin Structure of Enhancer-Deleted rRNA Genes in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (7) ◽  
pp. 4953-4960 ◽  
Author(s):  
Michael Banditt ◽  
Theo Koller ◽  
José M. Sogo
Keyword(s):  
Chromatin Structure ◽  
Transcriptional Activity ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Enhancer Element ◽  
Gel Retardation Assay ◽  
Gel Retardation ◽  
Gene Copies ◽  
Polymerase I

ABSTRACT We used the psoralen gel retardation assay and Northern blot analysis in an in vivo yeast system to analyze effects of rDNA enhancer deletions on the chromatin structure and the transcription of tagged rDNA units. We found that upon deletion of a single enhancer element, transcription of the upstream and downstream rRNA gene was reduced by about 50%. Although removing both flanking enhancers of an rRNA gene led to a further reduction in transcription levels, a significant amount of transcriptional activity remained, either resulting from the influence of more distantly located enhancer elements or reflecting the basal activity of the polymerase I promoter within the nucleolus. Despite the reduction of transcriptional activity upon enhancer deletion, the activation frequency (proportion of nonnucleosomal to nucleosomal gene copies in a given cell culture) of the tagged rRNA genes was not significantly altered, as determined by the psoralen gel retardation assay. This is a strong indication that, within the nucleolus, the yeast rDNA enhancer functions by increasing transcription rates of active rRNA genes and not by activating silent transcription units.

scholarly journals Chromatin structure and transcriptional activity around the replication forks arrested at the 3' end of the yeast rRNA genes.

1994 ◽  
Vol 14 (1) ◽  
pp. 318-326 ◽  
Author(s):  
R Lucchini ◽  
J M Sogo
Keyword(s):  
Chromatin Structure ◽  
Rrna Genes ◽  
Cross Linking ◽  
Rrna Gene ◽  
Replication Forks ◽  
Dna Encoding ◽  
Coding Region ◽  
Gene Copies ◽  
Inactive Genes

Replication intermediates containing forks arrested at the replication fork barrier near the 3' end of the yeast rRNA genes were analyzed at the chromatin level by using in vivo psoralen cross-linking as a probe for chromatin structure. These specific intermediates were purified from preparative two-dimensional agarose gels, and the extent of cross-linking in the different portions of the branched molecules was examined by electron microscopy and by using a psoralen gel retardation assay. The unreplicated section corresponding to the rRNA coding region upstream of the arrested forks appeared mostly heavily cross-linked, characteristic of transcriptionally active rRNA genes devoid of nucleosomes, whereas the replicated daughter strands representing newly synthesized spacer sequences showed a nucleosomal organization typical for bulk chromatin. The failure to detect replication forks arrested at the 3' end of inactive rRNA gene copies and the fact that most DNA encoding rRNA (rDNA) is replicated in the same direction as transcription suggest that replication forks seldom originate from origins of replication located immediately downstream of inactive genes.

scholarly journals Regulation of Ribosomal RNA Production by RNA Polymerase I: Does Elongation Come First?

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Benjamin Albert ◽  
Jorge Perez-Fernandez ◽  
Isabelle Léger-Silvestre ◽  
Olivier Gadal
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Chromatin Structure ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Elongation Factors ◽  
Pol I ◽  
Polymerase I

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structure in vivo. Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49 in vivo.

Chromatin structure and transcriptional activity around the replication forks arrested at the 3' end of the yeast rRNA genes

1994 ◽  
Vol 14 (1) ◽  
pp. 318-326
Author(s):  
R Lucchini ◽  
J M Sogo
Keyword(s):  
Chromatin Structure ◽  
Rrna Genes ◽  
Cross Linking ◽  
Rrna Gene ◽  
Replication Forks ◽  
Dna Encoding ◽  
Coding Region ◽  
Gene Copies ◽  
Inactive Genes

Replication intermediates containing forks arrested at the replication fork barrier near the 3' end of the yeast rRNA genes were analyzed at the chromatin level by using in vivo psoralen cross-linking as a probe for chromatin structure. These specific intermediates were purified from preparative two-dimensional agarose gels, and the extent of cross-linking in the different portions of the branched molecules was examined by electron microscopy and by using a psoralen gel retardation assay. The unreplicated section corresponding to the rRNA coding region upstream of the arrested forks appeared mostly heavily cross-linked, characteristic of transcriptionally active rRNA genes devoid of nucleosomes, whereas the replicated daughter strands representing newly synthesized spacer sequences showed a nucleosomal organization typical for bulk chromatin. The failure to detect replication forks arrested at the 3' end of inactive rRNA gene copies and the fact that most DNA encoding rRNA (rDNA) is replicated in the same direction as transcription suggest that replication forks seldom originate from origins of replication located immediately downstream of inactive genes.

scholarly journals Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes

1992 ◽  
Vol 12 (10) ◽  
pp. 4288-4296
Author(s):  
R Lucchini ◽  
J M Sogo
Keyword(s):  
Chromatin Structure ◽  
Rrna Genes ◽  
Cross Linking ◽  
Restriction Fragments ◽  
Coding Regions ◽  
Culture Cells ◽  
Gel Retardation Assay ◽  
Gene Copies ◽  
Frog Species ◽  
Active Genes

The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer.

scholarly journals Transcription in the yeast rRNA gene locus: distribution of the active gene copies and chromatin structure of their flanking regulatory sequences.

1995 ◽  
Vol 15 (10) ◽  
pp. 5294-5303 ◽  
Author(s):  
R Dammann ◽  
R Lucchini ◽  
T Koller ◽  
J M Sogo
Keyword(s):  
Chromatin Structure ◽  
Gene Locus ◽  
Transcription Unit ◽  
Rna Polymerase I ◽  
Yeast Cells ◽  
Rrna Gene ◽  
Open Chromatin ◽  
Gene Copies ◽  
Polymerase I ◽  
Rrna Gene Locus

In growing yeast cells, about half of the 150 tandemly repeated rRNA genes are transcriptionally active and devoid of nucleosomes. By using the intercalating drug psoralen as a tool to mark accessible sites along chromatin DNA in vivo, we found that the active rRNA gene copies are rather randomly distributed along the ribosomal rRNA gene locus. Moreover, results from the analysis of a single, tagged transcription unit in the tandem array are not consistent with the presence of a specific subset of active genes that is stably maintained throughout cell divisions. In the rRNA intergenic spacers of yeast cells, an enhancer is located at the 3' end of each transcription unit, 2 kb upstream of the next promoter. Analysis of the chromatin structure along the tandem array revealed a structural link between transcription units and adjacent, 3' flanking enhancer sequences: each transcriptionally active gene is flanked by a nonnucleosomal enhancer, whereas inactive, nucleosome-packed gene copies are followed by enhancers regularly packaged in nucleosomes. From the fact that nucleosome-free enhancers were also detected in an RNA polymerase I mutant strain, we interpret these open chromatin structures as being the result of specific protein-DNA interactions that can occur before the onset of transcription. In contrast, in this mutant strain, all of the rRNA coding sequences are packaged in nucleosomal arrays. This finding indicates that the establishment of the open chromatin conformation on the activated gene copies requires elongating RNA polymerase I molecules advancing through the template.

scholarly journals Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

1992 ◽  
Vol 12 (10) ◽  
pp. 4288-4296 ◽  
Author(s):  
R Lucchini ◽  
J M Sogo
Keyword(s):  
Chromatin Structure ◽  
Rrna Genes ◽  
Cross Linking ◽  
Restriction Fragments ◽  
Coding Regions ◽  
Culture Cells ◽  
Gel Retardation Assay ◽  
Gene Copies ◽  
Frog Species ◽  
Active Genes

The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer.

scholarly journals Deletion of Rnt1p Alters the Proportion of Open versus Closed rRNA Gene Repeats in Yeast

2007 ◽  
Vol 28 (2) ◽  
pp. 619-629 ◽  
Author(s):  
Mathieu Catala ◽  
Maxime Tremblay ◽  
Éric Samson ◽  
Antonio Conconi ◽  
Sherif Abou Elela
Keyword(s):  
Chromatin Structure ◽  
Ribosomal Proteins ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Open Chromatin ◽  
Rrna Processing ◽  
Rnase Iii ◽  
Double Stranded Rna ◽  
Open Versus ◽  
Polymerase I

ABSTRACT In Saccharomyces cerevisiae, the double-stranded-RNA-specific RNase III (Rnt1p) is required for the processing of pre-rRNA and coprecipitates with transcriptionally active rRNA gene repeats. Here we show that Rnt1p physically interacts with RNA polymerase I (RNAPI) and its deletion decreases the transcription of the rRNA gene and increases the number of rRNA genes with an open chromatin structure. In contrast, depletion of ribosomal proteins or factors that impair RNAPI termination did not increase the number of open rRNA gene repeats, suggesting that changes in the ratio of open and closed rRNA gene chromatin is not due to a nonspecific response to ribosome depletion or impaired termination. The results demonstrate that defects in pre-rRNA processing can influence the chromatin structure of the rRNA gene arrays and reveal links among the rRNA gene chromatin, transcription, and processing.

scholarly journals Expression of rRNA Genes and Nucleolus Formation at Ectopic Chromosomal Sites in the Yeast Saccharomyces cerevisiae

2006 ◽  
Vol 26 (16) ◽  
pp. 6223-6238 ◽  
Author(s):  
Melanie L. Oakes ◽  
Katsuki Johzuka ◽  
Loan Vu ◽  
Kristilyn Eliason ◽  
Masayasu Nomura
Keyword(s):  
The Other ◽  
Rrna Genes ◽  
Gene Copy ◽  
Rrna Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pol I ◽  
Gene Copies ◽  
Tandem Repetition ◽  
Polymerase I ◽  
Single Versus

ABSTRACT We constructed yeast strains in which rRNA gene repeats are integrated at ectopic sites in the presence or absence of the native nucleolus. At all three ectopic sites analyzed, near centromere CEN5, near the telomere of chromosome VI-R, and in middle of chromosome V-R (mid-V-R), a functional nucleolus was formed, and no difference in the expression of rRNA genes was observed. When two ribosomal DNA (rDNA) arrays are present, one native and the other ectopic, there is codominance in polymerase I (Pol I) transcription. We also examined the expression of a single rDNA repeat integrated into ectopic loci in strains with or without the native RDN1 locus. In a strain with reduced rRNA gene copies at RDN1 (∼40 copies), the expression of a single rRNA gene copy near the telomere was significantly reduced relative to the other ectopic sites, suggesting a less-efficient recruitment of the Pol I machinery from the RDN1 locus. In addition, we found a single rRNA gene at mid-V-R was as active as that within the 40-copy RDN1. Combined with the results of activity analysis of a single versus two tandem copies at CEN5, we conclude that tandem repetition is not required for efficient rRNA gene transcription.

scholarly journals A complex control region of the mouse rRNA gene directs accurate initiation by RNA polymerase I.

1985 ◽  
Vol 5 (3) ◽  
pp. 554-562 ◽  
Author(s):  
K G Miller ◽  
J Tower ◽  
B Sollner-Webb
Keyword(s):  
Initiation Site ◽  
Rrna Gene ◽  
Sufficient Information ◽  
Reaction Conditions ◽  
Cell Extracts ◽  
S 100 ◽  
Initiation Reaction ◽  
Polymerase I

To determine the size and location of the mouse rDNA promoter, we constructed systematic series of deletion mutants approaching the initiation site from the 5' and 3' directions. These templates were transcribed in vitro under various conditions with S-100 and whole-cell extracts. Surprisingly, the size of the rDNA region that determines the level of transcription differed markedly, depending on the reaction conditions. In both kinds of cell extracts, the apparent 5' border of the promoter was at residue ca. -27 under optimal transcription conditions, but as reaction conditions became less favorable, the 5' border moved progressively out to residues -35, -39, and -45. The complete promoter, however, extends considerably further, for under other nonoptimal conditions, we observed major effects of promoter domains extending in the 5' direction to positions ca. -100 and -140. In contrast, the apparent 3' border of the mouse rDNA promoter was at residue ca. +9 under all conditions examined. We also show that the subcloned rDNA region from -39 to +9 contains sufficient information to initiate accurately and that the region between +2 and +9 can influence the specificity of initiation. These data indicate that, although the polymerase I transcription factors recognize and accurately initiate with only the sequences downstream of residue -40, sequences extending out to residue -140 greatly favor the initiation reaction; presumably, this entire region is involved in rRNA transcription in vivo.

Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene

1987 ◽  
Vol 7 (1) ◽  
pp. 314-325
Author(s):  
C A Harrington ◽  
D M Chikaraishi
Keyword(s):  
Ribosomal Dna ◽  
Transcriptional Activity ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Rrna Synthesis ◽  
Rna Transcripts ◽  
Pair Sequence ◽  
Polymerase I

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

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