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A Krauskopf

Publications and source records attributed to A Krauskopf.

14 recordsLinked to original sources
PubMed2003

Mutant telomeres inhibit transcriptional silencing at native telomeres of the yeast Kluyveromyces lactis.

We report the identification and characterization of transcriptional silencing at native telomeres in the budding yeast Kluyveromyces lactis. We show that K. lactis telomeres are able to repress the transcription of a gene located at the junction between the telomeric repeat tract and the subtelomeric domain. As in Saccharomyces cerevisiae, switching between the repressed and derepressed transcriptional states occurs. C-terminal truncation of the telomere binding protein Rap1p, which leads to a regulated alteration in telomere length, reduces telomeric silencing. In addition, telomeric silencing is reduced dramatically in telomerase RNA mutants in which telomere length control has been lost. This is consistent with the possibility that the structure of the entire telomere affects the silencing functions exhibited by its internal domain.

Base Sequence↗
PubMed2001

Role for telomere cap structure in meiosis.

Telomeres, the natural ends of eukaryotic chromosomes, are essential for the protection of chromosomes from end-to-end fusions, recombination, and shortening. Here we explore their role in the process of meiotic division in the budding yeast, Kluyveromyces lactis. Telomerase RNA mutants that cause unusually long telomeres with deregulated structure led to severely defective meiosis. The severity of the meiotic phenotype of two mutants correlated with the degree of loss of binding of the telomere binding protein Rap1p. We show that telomere size and the extent of potential Rap1p binding to the entire telomere are irrelevant to the process of meiosis. Moreover, we demonstrate that extreme difference in telomere size between two homologous chromosomes is compatible with the normal function of telomeres during meiosis. In contrast, the structure of the most terminal telomeric repeats is critical for normal meiosis. Our results demonstrate that telomeres play a critical role during meiotic division and that their terminal cap structure is essential for this role.

Binding Sites↗
PubMed2000

Molecular manifestations and molecular determinants of telomere capping.

Multiple interacting components of the telomere, together with telomerase (and sometimes recombination), determine whether a telomere will be functional, allowing cell proliferation. The various components reinforce each other, providing for a robust and resilient system of protection and replenishment of telomeres. A characteristic of a telomere is that its structural features elicit responses that allow it to be dynamically recapped. Eliciting a DNA damage response through uncapping of a telomere appears to be one way in which telomerase action at that telomere is stimulated. Thus, as long as a timely and appropriate recapping of the telomere is possible, regulated uncapping of a telomere appears to be not only normal, but even required for optimal telomere maintenance. Telomere length and the presence of telomerase provide an example of a pair of interacting components that determine telomere capping function. Telomerase is dispensable in cells with sufficiently long telomeres; but in cells with short telomeres lacking telomerase, cells lose the ability to proliferate, and in some cell types, telomere fusions are increased. However, expressing telomerase can make even very short telomeres functional. Many interesting questions remain as to the mechanisms of these biological effects.

Animals↗
PubMed2000

Telomeres and their control.

Telomeres are DNA and protein structures that form complexes protecting the ends of chromosomes. Understanding of the mechanisms maintaining telomeres and insights into their function have advanced considerably in recent years. This review summarizes the currently known components of the telomere/telomerase functional complex, and focuses on how they act in the control of processes occurring at telomeres. These include processes acting on the telomeric DNA and on telomeric proteins. Key among them are DNA replication and elongation of one telomeric DNA strand by telomerase. In some situations, homologous recombination of telomeric and subtelomeric DNA is induced. All these processes act to replenish or restore telomeres. Conversely, degradative processes that shorten telomeric DNA, and nonhomologous end-joining of telomeric DNA, can lead to loss of telomere function and genomic instability. Hence they too must normally be tightly controlled.

Telomerase↗
PubMed1998

Rap1 protein regulates telomere turnover in yeast.

Telomere length is maintained through a dynamic balance between addition and loss of the terminal telomeric DNA. Normal telomere length regulation requires telomerase as well as a telomeric protein-DNA complex. Previous work has provided evidence that in the budding yeasts Kluyveromyces lactis and Saccharomyces cerevisiae, the telomeric double-stranded DNA binding protein Rap1p negatively regulates telomere length, in part by nucleating, by its C-terminal tail, a higher-order DNA binding protein complex that presumably limits access of telomerase to the chromosome end. Here we show that in K. lactis, truncating the Rap1p C-terminal tail (Rap1p-DeltaC mutant) accelerates telomeric repeat turnover in the distal region of the telomere. In addition, combining the rap1-DeltaC mutation with a telomerase template mutation (ter1-kpn), which directs the addition of mutated telomeric DNA repeats to telomeres, synergistically caused an immediate loss of telomere length regulation. Capping of the unregulated telomeres of these double mutants with functionally wild-type repeats restored telomere length control. We propose that the rate of terminal telomere turnover is controlled by Rap1p specifically through its interactions with the most distal telomeric repeats.

DNA, Fungal↗
PubMed1997

The telomere and telomerase: how do they interact?

The tandemly repeated DNA sequence of telomeres is typically specified by the ribonucleoprotein enzyme telomerase. Telomerase copies part of its intrinsic RNA moiety to make one strand of the telomeric repeat DNA. Recent work has led to the concept of a telomere homeostasis system. We have been studying two key physical components of this system: the telomere itself and telomerase. Mutating the template sequence of telomerase RNA caused various phenotypes: (1) mutating specific residues in the ciliate Tetrahymena and two yeasts showed that they are required for critical aspects of telomerase action; (2) certain mutated telomeric sequences caused a previously unreported phenotype, i.e. a strong anaphase block in Tetrahymena micronuclei; and (3) certain template mutations in the telomerase RNA gene of the yeast Kluyveromyces lactis led to unregulated telomere elongation, which in some cases was directly related to loss of binding to K. lactis Rap1p. Using K. lactis carrying alterations in the genes for Rap1p and other silencing components, we proposed a general model for telomere length homeostasis: namely, that the structure and DNA length of the DNA-protein complex that comprises the telomere are key determinants of telomerase access, and hence the frequency of action of telomerase, at the telomere.

Anaphase↗
PubMed1996

Control of telomere growth by interactions of RAP1 with the most distal telomeric repeats.

Telomeres, the specialized DNA-protein structures at the ends of eukaryotic chromosomes, are required for chromosomal stability and integrity. Regulation of the overall length of the telomeric DNA repeat tract is likely to be a key requirement for its various biological roles. We have studied telomere length regulation in the yeast Kluyveromyces lactis, which has long (25 base pairs) homogeneous telomeric repeat units that make it highly suitable for telomere studies. In the related Saccharomyces cerevisiae, the DNA-sequence-specific duplex-binding protein RAP1 is a component of the telomeric complex. Here we show that the phenotypic severity of previously described telomerase RNA (ter1) mutations is directly proportional to the loss of RAP1 binding to mutated telomeric repeats. Using a carboxy-terminal-tail mutant of K. lactis RAP1, we also show that, unexpectedly, RAP1 interaction with the most terminal telomeric repeats is crucial for telomere length control.

Base Sequence↗
PubMed1994

A cellular repressor regulates transcription initiation from the minute virus of mice P38 promoter.

We previously reported that the P38 promoter of minute virus of mice (MVM) is trans activated by the viral nonstructural protein, NS1, through an interaction with a downstream promoter element designated DPE. In this communication we report the identification of a distinct downstream promoter element which inhibits transcription from the P38 promoter in vitro, in the absence of the DPE. Removal of 34 bp from the region between +95 and +129 downstream from the P38 initiation start site relieved inhibition of transcription in whole-cell extract. Inhibition was also relieved by the addition, to the transcription reaction, of excess DNA fragments which span the putative inhibiting element. This indicated the involvement of a trans-acting factor, in inhibition of transcription from the P38. Gel retardation experiments demonstrated the specific binding of a cellular protein to the inhibitory element. This P38 inhibitory element shows spacing and orientation dependence as well as promoter specificity. The regulation of viral transcription by a cellular repressor may play an important role in obtaining a fine temporal order of viral gene expression during the course of infection.

Base Sequence↗
PubMed1994

Minute virus of mice infection modifies cellular transcription elongation.

Our previous observations indicated that upon infection with minute virus of mice (MVM), Ehrlich ascites cells lose a transcription elongation activity which is essential for the readthrough of the MVM attenuator. This was monitored by the ability of extracts from uninfected but not from infected cells to support readthrough of the P4 attenuator when added to partially purified transcription elongation complexes. We have investigated the nature of this change in transcription elongation following MVM infection. In this communication, we show that infection of Ehrlich ascites cells with MVM leads to a general shift in the length of nascent mRNA synthesized in isolated nuclei and separated by sucrose gradients. Furthermore, infection leads to attenuation of transcription of the cellular gene c-fos but not c-myc. We show biochemical evidence to support a model by which, following MVM infection, there is a functional reduction in the activity of a TFIIS-like general transcriptional elongation activity.

Animals↗
PubMed1993

Wild-type but not mutant p53 can repress transcription initiation in vitro by interfering with the binding of basal transcription factors to the TATA motif.

It has previously been shown that excess wild type (wt) p53 can repress the transcriptional activity of a variety of promoters in intact cells. To determine whether this transcriptional repression represented a direct effect of p53, wt and mutant p53 were prepared from E. coli-produced p53 and from insect cells infected with a recombinant baculovirus. When added into an in vitro transcription system, wt p53, but not mutant p53 reduced markedly transcription from the c-myc promoter, as well as from an array of other promoters, with the exception of an MHC class I gene promoter. The presence of wt p53 seemed to affect specifically the formation of the transcription preinitiation complex because preformed initiation complexes were completely refractory to wt p53, as was also the process of transcript elongation. Wild-type but not mutant p53 interfered with the stable binding of TBP and TFIIA to the TATA motif, although both wt and mutant p53 could associate in vitro with purified TBP. We propose that upon binding to TBP, wt but not mutant p53 specifically blocks the ability of TBP to engage in interactions required for efficient transcriptional initiation. This may account, at least in part, for the ability of excess wt p53 to inhibit cell proliferation and to interfere with neoplastic processes.

Base Sequence↗
PubMed1991

Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II.

We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II.

Adenoviruses, Human↗
PubMed1991

The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.

We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and IIX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.

Animals↗
PubMed1990

A cis downstream element participates in regulation of in vitro transcription initiation from the P38 promoter of minute virus of mice.

We report the use of a HeLa whole cell extract (WCE) runoff transcription system for the study of cis- and trans-acting elements, participating in the regulation of transcription initiation from the P38 promoter of the parvovirus minute virus of mice (MVM). Our initial studies with HeLa WCE indicated that transcription from the P38 promoter is very inefficient, compared with transcription from the P4 promoter. Supplementation of the HeLa WCE with WCE prepared from uninfected Ehrlich ascites cells enhanced transcription from the P38 promoter twofold, indicating a role for a cellular factor in transcription from the P38 promoter. Furthermore, supplementation with WCE prepared from MVM-infected Ehrlich ascites cells enhanced transcription from the P38 promoter about sixfold, indicating a role for a virally encoded or induced factor. Analyses of runoffs produced by transcription of DNA templates digested with various restriction enzymes defined a downstream promoter element (DPE) necessary for efficient transcription initiation from the P38 promoter. This element resides 282 to 647 base pairs 3' to the transcription initiation site, between the NarI site and the HindIII site (2287 to 2652, MVM numbering system). The virally encoded NS1 protein was shown by DNA precipitation to bind directly or indirectly through a cellular factor to the DPE. This interaction is suggested to be involved in the up regulation of the P38 promoter of MVM. Finally, with a DNase I protection assay performed on a fragment containing the DPE, we estimated the sequence involved in the binding of a factor present in uninfected and infected extracts. The correlation between the binding and transcription activation is discussed.

Animals↗