Molecular analysis of the heterogeneity region of the human ribosomal spacer

Volpe, Adriana La; Simeone, Antonio; D’Esposito, Maurizio; Scotto, Luigi; Fidanza, Vincenzo; Falco, Antonietta de; Boncinelli, Edoardo

doi:10.1016/0022-2836(85)90214-1

Cited by 68 publications

(38 citation statements)

References 34 publications

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“…Since Labhart and Reeder (27) have reported that the event 265 nucleotides downstream of the 3' end of 28S rRNA in X. laevi.s (which they believe to be processing) is completely inhibited by heat shock, we tested whether heat or other stresses might have a similar effect in human cells. [29], whose nucleotide +350 corresponds to +362 on our sequence, which was kindly provided by J. Sylvester and R. Schmickel). Using a probe that extended from +218 to either +384 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Parker¹,

Bond²

1989

Mol. Cell. Biol.

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Human rRNA precursors from normal or stressed HeLa cells were studied by Si nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with[32p]PO4. Heating cells to 43°C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42°C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43°C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

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Section: Discussionmentioning

confidence: 99%

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Parker¹,

Bond²

1989

Mol. Cell. Biol.

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“…crystal structure | RNA polymerase I | transcription termination | protein-DNA interaction | replication termination E ukaryotic rDNAs are found as multiple tandem copies encoding pre-rRNA and upstream and downstream regulatory elements (1-3) including DNA sequences (Ter) that promote site-specific termination of transcription catalyzed by RNA polymerase I (pol I) from yeast to humans (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Specialized transcription terminator proteins bind to Ter sites and not only arrest transcription by RNA polymerase I (pol I) in a polar mode but also replication forks approaching from the opposite direction.…”

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confidence: 99%

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe

Jaiswal

Choudhury

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Reb1 of Schizosaccharomyces pombe represents a family of multifunctional proteins that bind to specific terminator sites (Ter) and cause polar termination of transcription catalyzed by RNA polymerase I (pol I) and arrest of replication forks approaching the Ter sites from the opposite direction. However, it remains to be investigated whether the same mechanism causes arrest of both DNA transactions. Here, we present the structure of Reb1 as a complex with a Ter site at a resolution of 2.7 Å. Structure-guided molecular genetic analyses revealed that it has distinct and well-defined DNA binding and transcription termination (TTD) domains. The region of the protein involved in replication termination is distinct from the TTD. Mechanistically, the data support the conclusion that transcription termination is not caused by just high affinity Reb1-Ter protein-DNA interactions. Rather, protein-protein interactions between the TTD with the Rpa12 subunit of RNA pol I seem to be an integral part of the mechanism. This conclusion is further supported by the observation that double mutations in TTD that abolished its interaction with Rpa12 also greatly reduced transcription termination thereby revealing a conduit for functional communications between RNA pol I and the terminator protein.crystal structure | RNA polymerase I | transcription termination | protein-DNA interaction | replication termination E ukaryotic rDNAs are found as multiple tandem copies encoding pre-rRNA and upstream and downstream regulatory elements (1-3) including DNA sequences (Ter) that promote site-specific termination of transcription catalyzed by RNA polymerase I (pol I) from yeast to humans (4-14). Specialized transcription terminator proteins bind to Ter sites and not only arrest transcription by RNA polymerase I (pol I) in a polar mode but also replication forks approaching from the opposite direction. Several studies have suggested that pol I transcription termination is a multistep process that requires (i) pausing of chain elongation by the terminator protein and (ii) dissociation and release of pol I and the primary transcript from the template. In mice, dissociation of pol I and release of the transcript require the release factor PTRF, a 44-kDa protein that interacts with the largest subunit of pol I (15). In yeast, additional factors for the processing of the end include the endonuclease Rnt1, the 5′-3′ Rat1 exonuclease, Sen1 helicase, and the kinase Grc3. They are part of an alternate pathway for termination by transcriptional coprocessing (4,(16)(17)(18)(19). In addition, in vivo analyses of Saccharomyces cerevisiae have shown a requirement for Rpa12, a component of pol I necessary for transcription termination (20). Whether the polar arrest of transcription is caused by an interaction between Rpa12 with the terminator protein is unknown.The terminator proteins that mediate transcription termination have been identified in multiple organisms and include Reb1 and Nsi1 [also called yeast transcription terminator (Ytt1)] of S. cerevisiae (...

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“…Each of these DNA modules contains several Sall restriction sites. Figure 1 shows the nucleotide sequences surrounding the five SalI sites present in the smallest class of cloned spacer fragments as determined by La Volpe et al (14). Interestingly, 10 nucleotides are highly conserved, eight of which exert a dyad symmetry and are shared by the mouse Sall box consensus sequence.…”

Section: Resultsmentioning

confidence: 99%

“…Individual human ribosomal transcription units exert a characteristic length variation within a specific region downstream from the 3' end of the transcribed region (4,12,14,17). The nucleotide sequence of this heterogeneous region has been determined for three different size classes and has been shown to be generated by multiple duplications of a fundamental DNA module of 700 to 800 bp (14). Each of these DNA modules contains several Sall restriction sites.…”

Section: Resultsmentioning

confidence: 99%

“…This microheterogeneity in the human 3'-terminal NTS region consists of essentially four fragment length variants that apparently were generated by duplication of two discrete sequence modules of about 700 and 800 base pairs (bp), respectively. Interestingly, this length variation is confined within some hundreds of base pairs of spacer sequences starting at a Sall restriction site at position +365 with respect to the 28S rRNA terminus and includes some closely spaced Sall sites (14). These clustered SalT restriction sites are part of a larger conserved sequence element, TCCcN0,3GGGTCGACC', which is present at least five times in the smallest size variant of the human 3' spacer.…”

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confidence: 99%

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Evolutionary changes of sequences and factors that direct transcription termination of human and mouse ribsomal genes.

Bartsch¹,

Schoneberg²,

Grummt³

1987

Mol. Cell. Biol.

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We have analyzed the sequences required for termination of human rDNA transcription. The human ribosomal transcription unit is shown to extend about 350 nucleotides into the 3'-terminal spacer and ends immediately upstream of a region with a distinct sequence heterogeneity. This heterogeneous region contains a cluster of conserved 10-base pair sequence elements which exert a striking homology to the proximal part of the 18-base pair murine rDNA transcription termination signal sequence, termed Sall box. Exonuclease IH protection assays and in vitro transcription experiments with both homologous and heterologous human-mouse minigene constructs, and extracts from HeLa or Ehrlich ascites cells, reveal a functional analogy of the human sequence to the mouse Sail box. It mediates binding of a nuclear protein which functions as a transcription termination factor. The murine signal sequence is recognized by the human factor but not vice versa. The different sequence specificities and electrophoretic properties of the functionally equivalent protein factors suggest that a molecular coevolution has taken place between the termination signal sequences and the genes coding for the termination factors.

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Molecular analysis of the heterogeneity region of the human ribosomal spacer

Cited by 68 publications

References 34 publications

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe

Evolutionary changes of sequences and factors that direct transcription termination of human and mouse ribsomal genes.

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