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R H Reeder

Publications and source records attributed to R H Reeder.

At least 19 recordsLinked to original sources
PubMed1992

Variants of the TATA-binding protein can distinguish subsets of RNA polymerase I, II, and III promoters.

Transcription extracts prepared from yeast that are deficient in the TATA-binding protein (TBP or TFIID) are also impaired in specific promoter recognition by all three nuclear RNA polymerases (pol I, II, and III). Specific initiation can be rescued by the addition of purified recombinant TBP, demonstrating that pol I, II, and III all require this factor. A mutation of TBP has been identified that will function with pol I but not with pol II or III. Conversely, another mutation, which inactivates TATA element binding in vitro, will function with pol I and III promoters but is inactive for a pol II promoter. Thus, it is possible to identify TBP variants that will only function on different subsets of all nuclear promoters.

DNA-Binding Proteins↗
PubMed1992

In vitro definition of the yeast RNA polymerase I promoter.

The structure of the ribosomal gene promoter from Saccharomyces cerevisiae has been analyzed in a whole cell in vitro extract. The promoter contains at least two essential domains, an upstream domain located at the 5' boundary near position -150 and a core promoter domain around the site of transcription initiation at +1. The upstream domain augments transcription in vitro but is not absolutely required. Maintenance of correct spacing between the two domains is critical. The in vitro analysis agrees well with prior in vivo analysis and it appears that the yeast promoter has a structure very similar to that of vertebrate ribosomal gene promoters.

Base Sequence↗
PubMed1992

Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation.

Previous work has shown that rRNA synthesis is strongly inhibited in yeast top1-top2 double mutants. Here, we show that inactivation of yeast topoisomerases can have paradoxical effects on transcription by RNA polymerase I. For example, transcription of ribosomal minigenes on extrachromosomal plasmids is greatly stimulated in top1-top2 cells while accumulation of full-length endogenous rRNA is strongly inhibited. We present evidence for a mechanism that can partly account for these opposing effects on transcription. On the one hand, transcription initiation can be stimulated owing to an accumulation of negative superhelicity because polymerase I prefers to initiate on negatively supercoiled templates. Conversely, synthesis of full-length rRNA is inhibited owing to the fact that chain elongation requires a DNA relaxing activity.

Cloning, Molecular↗
PubMed1992

CPT Codes for 1992.

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Abstracting and Indexing↗
PubMed1991

Specific initiation by RNA polymerase I in a whole-cell extract from yeast.

A protocol is described for making a soluble whole-cell extract from yeast (Saccharomyces cerevisiae) that supports active and specific transcription initiation by RNA polymerases I, II, and III. Specific initiation by polymerase I decreases in high-density cultures, paralleling the decrease in abundance of the endogenous 35S rRNA precursor. This extract should be useful for studying the molecular mechanisms that regulate rRNA transcription in yeast.

Base Sequence↗
PubMed1991

xUBF and Rib 1 are both required for formation of a stable polymerase I promoter complex in X. laevis.

We show that three protein fractions are required for accurate transcription initiation at a Xenopus laevis ribosomal gene promoter in vitro: RNA polymerase I, Rib1 and xUBF. The Rib1 and xUBF fractions are both necessary and sufficient for formation of a stable initiation complex. The xUBF fraction can be completely replaced by recombinant xUBF. We also report the sequence of a cDNA clone for xUBF. xUBF is 701 amino acids in length, contains domain which are related to a domain found in chromosomal proteins HMG 1 and 2, and has an acidic carboxy terminus of 87 amino acids. xUBF is closely similar in amino acid sequence to its previously reported human homolog, hUBF, except that xUBF has only three of the HMG-related domains while hUBF has four and therefore is 63 amino acids longer than xUBF.

Amino Acid Sequence↗
PubMed1991

xUBF contains a novel dimerization domain essential for RNA polymerase I transcription.

Xenopus laevis upstream binding factor (xUBF) is an RNA polymerase I transcription factor that is required for formation of the stable initiation complex. The 701-amino-acid protein contains three regions of homology to the chromosomal protein HMG1 (the HMG boxes), which act in comparative independence to cause DNA binding. DNA binding is augmented by a 102-residue amino-terminal domain that causes xUBF to form dimers. The dimerization domain is bipartite in structure, consisting of two regions with the potential to form amphipathic helices, separated by a gap of at least 22 amino acids. The carboxyl half of xUBF is relatively dispensable for transcription (including an 87-residue acidic tail). However, either altering the number of HMG boxes or interfering with dimerization eliminates transcription. The gap region of the dimerization domain is dispensable for dimerization but is absolutely required for transcription. This suggests that the gap region has a critical function in transcription distinct from any effect on dimerization or DNA binding.

Amino Acid Sequence↗
PubMed1990

Functional difference between the sites of ribosomal 40S precursor 3' end formation in Xenopus laevis and Xenopus borealis.

In the ribosomal genes of X. laevis, the sequence GACTTGCNC is found about 60bp upstream of the gene promoter (T3) and is necessary and sufficient to cause termination of RNA polymerase I transcription. At the 3' end of the 40S precursor coding region (T2) a sequence differing by one nucleotide, GACTTGCNG, directs RNA 3' end formation but allows polymerase to transcribe on into the intergenic spacer (Labhart and Reeder, 1989, Genes and Dev. 4: 269-276). Sites corresponding to T2 and T3 are also found in a related species, X. borealis. Inspection of the T2 sequence in X. borealis reveals that it contains two copies of the terminator sequence, GACTTGCNC, located 15 and 96 bp downstream of the 3' end of the 40S precursor coding region. Here we present functional tests of those two T2 elements that show that, as predicted from the sequence, they both show termination activity and are functionally indistinguishable from the T3 site in X. laevis. These results suggest that X. laevis T2 is an example of a naturally occurring point mutation, and the inability to terminate transcription at T2 is an exception to the general pattern of ribosomal gene transcription in higher eukaryotes.

Animals↗
PubMed1990

rRNA synthesis in the nucleolus.

The past year has seen advances in our understanding of three broad areas that concern ribosomal RNA production. It is becoming apparent that for a large number of eukaryotes, sequence elements that regulate ribosomal RNA transcription are arranged in a similar pattern. This conservation of arrangement implies conservation of regulatory mechanisms. Better understanding of the ribosomal gene transcription factors has emerged, and one factor has been purified and cloned. In vitro systems for processing ribosomal RNA are beginning to be developed, allowing the first direct proof that a small nuclear ribonucleoprotein (U3) is involved in ribosomal RNA processing.

Animals↗
PubMed1990

A point mutation uncouples RNA 3'-end formation and termination during ribosomal gene transcription in Xenopus laevis.

Two sites, T2 and T3, in the ribosomal gene spacer of Xenopus laevis both direct RNA 3'-end formation 15 bp upstream of the conserved box sequence GACTTGC. Site T2, which defines the 3' end of the 40S precursor, does not terminate transcription whereas site T3 at the 3' end of the spacer does. Here we show that T2 can be converted into a T3-like site with termination activity by a single point mutation 2 bp downstream of the T2 box. RNA 3'-end formation at T2 is unchanged by this mutation. Conversely, a point mutation 2 bp downstream of the T3 box inhibits termination without affecting 3'-end formation. Our results identify two separable events occurring at the 3' end of the ribosomal genes: (1) RNA 3'-end formation by processing and (2) transcription termination. The two processes are directed by two distinct, but overlapping, signals in the DNA sequence. Site T2 in X. laevis is damaged in the second process by a natural mutation.

Animals↗
PubMed1990

An RNA polymerase I termination site can stimulate the adjacent ribosomal gene promoter by two distinct mechanisms in Xenopus laevis.

On the ribosomal genes of Xenopus laevis, the T3 terminator is located approximately 60 bp upstream of the 5' boundary of the gene promoter. We have shown previously that mutation of the terminator simultaneously abolishes termination and impairs initiation by RNA polymerase I. Here, we show that the terminator influences the promoter by two distinct mechanisms. In one mechanism the terminator protects the promoter by preventing polymerase from reading through the initiation complex. In a second mechanism, the terminator interacts directly with the promoter, whether or not termination occurs. This positive interaction requires precise positioning of the terminator relative to the promoter and is sensitive to movement of the terminator by as little as 1 or 2 bp. We conclude that the terminator and promoter interact as one interdependent complex.

Animals↗
PubMed1990

rUBF, an RNA polymerase I transcription factor from rats, produces DNase I footprints identical to those produced by xUBF, its homolog from frogs.

Rat cells contain a DNA-binding polymerase I transcription factor, rUBF, with properties similar to UBF homologs that have been purified from both human (hUBF) and frog (xUBF) cells. In this note we report the affinity purification of rUBF to apparent homogeneity and show that UBFs from both rat and frog have identical footprinting characteristics on templates from either species. Furthermore, xUBF was able to stimulate transcription from rat RNA polymerase I promoters in a partially fractionated rat extract that was UBF dependent. These results strengthen the conclusion that all vertebrate cells contain a UBF homolog whose DNA-binding specificity and function have been strongly conserved.

Animals↗
PubMed1990

Enhancers for RNA polymerase I in mouse ribosomal DNA.

The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.

Animals↗
PubMed1989

Molecular mechanisms governing species-specific transcription of ribosomal RNA.

An unusual property of ribosomal RNA transcription is the species specificity of promoter recognition. Unexpectedly, the sequence-specific RNA pol I transcription factors hUBF and xUBF, isolated from human and Xenopus cells, respectively, recognize the same DNA sequence elements. Despite this similarity in DNA binding activity, neither factor will functionally substitute for the other in reconstituted transcription assays, suggesting that the specificity of protein-DNA interactions cannot account for the species-specific activation of transcription by hUBF and xUBF. Interestingly, we find that hUBF and xUBF form distinctly different complexes with human SL1 at both the human and Xenopus promoters. Together these results strongly implicate specific protein-protein interactions between transcription factors as an important determinant of promoter selectivity and species specificity.

Animals↗
PubMed1989

A test of 'polymerase handover' as a mechanism for stimulating initiation by RNA polymerase I.

On the tandemly linked ribosomal genes of Xenopus laevis, the RNA polymerase transcribes past the 3' end of the 40S coding region and terminates at T3 just upstream of the gene promoter. The close proximity of T3 to the gene promoter, and the functional interdependence of these two elements, has led to the suggestion that polymerase terminating at T3 might be passed directly to the gene promoter. Such a mechanism might be necessary to maintain the characteristic high rate of transcription initiation seen on the ribosomal genes. We have performed a direct test of this model by introducing chain-terminating psoralen adducts into a circular plasmid containing a single gene promoter with its attendant T3 region upstream. We find that the psoralen adducts can completely prevent polymerase from traveling around the template circle (and thus prevent polymerase from approaching the promoter from upstream) without affecting the rate of transcription initiation at the gene promoter. This result suggests that recycling of polymerase from T3 to the promoter is not a significant mechanism in maintaining high initiation rates.

Animals↗
PubMed1989

High initiation rates at the ribosomal gene promoter do not depend upon spacer transcription.

We report experiments that test the model that in Xenopus laevis, RNA polymerase I is "handed over" in a conservative fashion from the T3 terminator to the adjacent gene promoter. We have introduced transcription-terminating lesions into the ribosomal DNA repeat by irradiating cultured cells with ultraviolet light. We used isolated nuclei to measure the effect of such lesions on transcription. UV damage sufficient to prevent all elongating RNA polymerase from reaching T3 from upstream had no adverse effect on the density of RNA polymerase at the very 5' end of the gene. We conclude that high rates of transcription initiation at the gene promoter do not depend upon polymerase passing from one repeat to the next or on polymerase initiating at the spacer promoters.

Animals↗