An apparent pause site in the transcription unit of the rabbit α-globin gene

Vandenbergh, David J.; James-Pederson, Magdalena; Hardison, Ross C.

doi:10.1016/0022-2836(91)90011-t

Cited by 13 publications

(8 citation statements)

References 54 publications

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“…The immediate consequence of signaling need not be transcript release as is generally assumed. It could be pausing, consistent with the apparent collection of paused transcription complexes seen in the striking electron micrographs of Osheim et al (61) and as suggested by the broad pausing profiles commonly seen downstream of poly(A) sites following nascent transcript run-on analysis (7,13,16,33,41,53,74,78). We have therefore designed an assay that measures the collective effects of the poly(A) signal on the efficiency of transcription elongation.…”

supporting

confidence: 73%

“…Pausing of the polymerase is thought to be necessary to allow the poly(A) site time to act and, thereby, to signal the polymerase. Consistent with this hypothesis pausing is indeed frequently observed to occur downstream of poly(A) sites (7,13,16,33,41,53,74,78). Also consistent with this hypothesis, some poly(A) sites are known to be accompanied by downstream elements that enhance cleavage and polyadenylation and/or termination or arrest (2,6,13,21,31,33,72).…”

supporting

confidence: 53%

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Mechanism of Poly(A) Signal Transduction to RNA Polymerase II In Vitro

Tran

Kim

Park

et al. 2001

Molecular and Cellular Biology

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Termination of transcription by RNA polymerase II usually requires the presence of a functional poly(A) site. How the poly(A) site signals its presence to the polymerase is unknown. All models assume that the signal is generated after the poly(A) site has been extruded from the polymerase, but this has never been tested experimentally. It is also widely accepted that a "pause" element in the DNA stops the polymerase and that cleavage at the poly(A) site then signals termination. These ideas also have never been tested. The lack of any direct tests of the poly(A) signaling mechanism reflects a lack of success in reproducing the poly(A) signaling phenomenon in vitro. Here we describe a cell-free transcription elongation assay that faithfully recapitulates poly(A) signaling in a crude nuclear extract. The assay requires the use of citrate, an inhibitor of RNA polymerase II carboxyl-terminal domain phosphorylation. Using this assay we show the following. It has become clear in recent years that RNA polymerase II (RNAPII) not only transcribes the mRNA but shepherds it through the stages of processing as well (42, 67). An early indicator of this coupling between transcription and processing was the finding that the poly(A) signal for cleavage and polyadenylation of pre-mRNA directs not only 3Ј-end processing of the transcript but also termination of transcription by the polymerase (20,32,50,61,77,79). A major challenge has been to explain how the poly(A) signal communicates with the polymerase.The core poly(A) signal in vertebrates consists of two recognition elements flanking a cleavage-polyadenylation site (76,82). Typically, an almost invariant AAUAAA hexamer lies 20 to 50 nucleotides (nt) upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5Ј cleavage product. The cleavage is mediated in vitro by a large, multicomponent protein complex that can be separated into five distinguishable factors. Two of these factors are the cleavage and polyadenylation specificity factor (CPSF), which binds the AAUAAA motif, and the cleavage stimulation factor (CstF), which binds the downstream U-rich element. In vitro studies suggest that CPSF (26) and probably CstF as well (55, 71) are recruited to the polymerase at the promoter. Presumably they then ride with the polymerase during transcription, scanning the extruding transcript so as to snare the poly(A) site when it emerges. The situation in yeast may be similar (8,29). Strictly speaking, the term "poly(A) site" refers only to the point at which cleavage occurs and the poly(A) tail is appended, but we use the term here to refer to the poly(A) signal as a whole when this helps to distinguish between the poly(A) signal as an entity and poly(A) signal transduction [or "poly(A) signaling"] as a process.Models that attempt to explain transduction of the signal from the poly(A) site to the polymerase can be divided into two categories (50): (i) cl...

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supporting

confidence: 73%

supporting

confidence: 53%

Mechanism of Poly(A) Signal Transduction to RNA Polymerase II In Vitro

Tran

Kim

Park

et al. 2001

Molecular and Cellular Biology

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“…Often, polymerase density decreases gradually downstream of the poly(A) site, as assessed by run-on transcription (20 -27). In many instances this decrease in polymerase density is preceded by a region in which the polymerase density is higher than it is over the body of the gene (25)(26)(27)(28)(29)(30)(31)(32)(33). This has generally been interpreted to indicate the presence of polymerase pausing in the region, but the basis for this pausing is not understood.…”

mentioning

confidence: 99%

The Poly(A) Signal, without the Assistance of Any Downstream Element, Directs RNA Polymerase II to Pause in Vivo and Then to Release Stochastically from the Template

Orozco

Kim

Martinson

2002

Journal of Biological Chemistry

114

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Genes encoding polyadenylated mRNAs depend on their poly(A) signals for termination of transcription. Typically, transcription downstream of the poly(A) signal gradually declines to zero, but often there is a transient increase in polymerase density immediately preceding the decline. Special elements called pause sites are traditionally invoked to account for this increase. Using run-on transcription from the nuclei of transfected cells, we show that both the pause and the gradual decline that follow a poly(A) site are generated entirely by the poly(A) signal itself in a series of model constructs. We found no other elements to be involved and argue that the elements called pause sites do not function through pausing. Both the poly(A)-dependent pause and the subsequent decline occurred earlier for a stronger poly(A) signal than for a weaker one. Because the gradual decline resembles the abortive elongation that occurs downstream of many promoters, one model has proposed that the poly(A) signal flips the polymerase from the elongation mode to the abortive mode like a binary switch. We compared abortive elongators with poly(A) terminators and found a 4-fold difference in processivity. We conclude that poly(A) terminating polymerases do not merely revert to their prior state of low processivity but rather convert to a new terminationprone condition.

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“…This ␣-like globin gene retains a CpG island (19) but does not encode a known, functional globin (11). No detectable transcription from this CpG island was observed in cellfree assays (11), in nuclear run-on assays in rabbit fetal liver cells (44), or in stably transfected K562 cells (data not shown). It lacks the 5Ј flanking and intragenic protein binding sites seen in the ␣-globin CpG island due to numerous point mutations and sequence replacements.…”

Section: Resultsmentioning

confidence: 96%

CpG Islands from the α-Globin Gene Cluster Increase Gene Expression in an Integration-Dependent Manner

Shewchuk

Hardison

1997

Molecular and Cellular Biology

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In contrast to other globin genes, the human and rabbit ␣-globin genes are expressed in transfected erythroid and nonerythroid cells in the absence of an enhancer. This enhancer-independent expression of the ␣-globin gene requires extensive sequences not only from the 5 flanking sequence but also from the intragenic region. However, the features of these internal sequences that are responsible for their positive effect are unclear. We tested several possible determinants of this activity. One possibility is that a previously identified array of discrete binding sites for known and potential regulatory proteins within the ␣-globin gene comprise an intragenic enhancer specific for the ␣-globin promoter, but directed rearrangements of the sequences show that this is not the case. Alternatively, the promoter may extend into the gene, with the function of the discrete binding sites being dependent on maintenance of their proper positions and orientations relative to the 5 flanking sequence. However, the positive effects observed in gene fusions do not localize to a discrete region of the ␣-globin gene and the results of internal deletions and point mutations argue against a required role of the targeted discrete binding sites. A third possibility is that the CpG island, which includes both the 5 flanking and intragenic regions associated with the positive activity, may itself have a more general effect on expression in transfected cells. Indeed, we show that the size of the CpG island in constructs correlates with the level of gene expression. Furthermore, the ␣-globin promoter is more active in the context of a previously inactive CpG island than in an A؉T-rich context, showing that the CpG island provides an environment more permissive for expression. These effects are seen only after integration, suggesting a possible mechanism at the level of chromatin structure.

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An apparent pause site in the transcription unit of the rabbit α-globin gene

Cited by 13 publications

References 54 publications

Mechanism of Poly(A) Signal Transduction to RNA Polymerase II In Vitro

Mechanism of Poly(A) Signal Transduction to RNA Polymerase II In Vitro

The Poly(A) Signal, without the Assistance of Any Downstream Element, Directs RNA Polymerase II to Pause in Vivo and Then to Release Stochastically from the Template

CpG Islands from the α-Globin Gene Cluster Increase Gene Expression in an Integration-Dependent Manner

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