The Xenopus laevis U2 gene distal sequence element (enhancer) is composed of four subdomains that can act independently and are partly functionally redundant.

The sequences involved in enhancement of transcription of the Xenopus U2 small nuclear RNA gene by the distal sequence element (DSE) of its promoter were analyzed in detail by microinjection of mutant genes into Xenopus oocytes. The DSE was shown to be roughly 60 base pairs long. Within this region, four motifs were found to contribute to DSE function: an ATGCAAAT octamer sequence, an SpI binding site, and two additional motifs which, since they are related in sequence, may bind the same transcription factor. These motifs were named D2 (for DSE; U2). Both the octamer sequence and the SpI site bound nuclear factors in vitro, but no factor binding to the D2 motifs was detected. All four elements were independently capable of enhancing transcription of the U2 gene to some extent. Furthermore, when assayed under both competitive and noncompetitive conditions, the individual units of the DSE displayed functional redundancy.

CDK6-a review of the past and a glimpse into the future: from cell-cycle control to transcriptional regulation.

The G1 cell-cycle kinase CDK6 has long been thought of as a redundant homolog of CDK4. Although the two kinases have very similar roles in cell-cycle progression, it has recently become apparent that they differ in tissue-specific functions and contribute differently to tumor development. CDK6 is directly involved in transcription in tumor cells and in hematopoietic stem cells. These functions point to a role of CDK6 in tissue homeostasis and differentiation that is partially independent of CDK6's kinase activity and is not shared with CDK4. We review the literature on the contribution of CDK6 to transcription in an attempt to link the new findings on CDK6's transcriptional activity to cell-cycle progression. Finally, we note that anticancer therapies based on the inhibition of CDK6 kinase activity fail to take into account its kinase-independent role in tumor development.

A factor with Sp1 DNA-binding specificity stimulates Xenopus U6 snRNA in vivo transcription by RNA polymerase III.

We have previously shown that transcription of the Xenopus U6 snRNA gene by RNA polymerase III is stimulated in injected Xenopus oocytes by an activator element termed the DSE, which contains an octamer sequence. Data presented here reveal that the DSE contains, in addition, a GC-rich sequence capable of binding Sp1. Both elements are required to obtain wild-type levels of U6 transcription in vivo. The Xenopus U6 DSE exhibits optimal activation properties only when positioned at its normal location upstream from the start site. The U6 Sp1 motif binds the mammalian Sp1 transcriptional activator independently of the Oct-1 protein in vitro. Those mutations that lead to a reduced transcription level in vivo abolish the binding of Sp1 in vitro. Thus, transcriptional stimulation through the Xenopus U6 Sp1 motif is likely to be mediated by a protein with DNA-binding specificity identical to mammalian Sp1. These findings support the notion that RNA polymerase II and III transcription complexes share transactivators.

The molecular basis of kirromycin (mocimycin) action; a 1H NMR study using deuterated elongation factor Tu.

The binding of the antibiotic kirromycin (mocimycin) to its target protein, bacterial elongation factor Tu (EF-Tu), has been studied by 1H NMR spectroscopy using deuterated protein. Narrow lines were observed in the spectrum of the unbound protein (due to residual protons) and in the spectrum of the kirromycin-EF-Tu complex. The spectrum of the complex has been compared with the spectra of the unbound protein and the unbound drug, and the results are interpreted in terms of the mode of antibiotic action of kirromycin.

Positionally exact initiation is required for the formation of a stable RNA polymerase II transcription complex in vivo.

The requirements for the formation of a stable transcription complex on the RNA polymerase II-transcribed Xenopus U2 snRNA gene have been analysed in vivo by oocyte microinjection experiments. The two elements of the U2 promoter which are located in the 5' flanking region of the gene, the DSE and the PSE, are shown to be essential but not sufficient for stable complex formation. Two additional elements are required. The first is a short gene-internal sequence; the second is the nucleotide at the normal point of initiation, which must be a purine. If this nucleotide is changed to a pyrimidine the site of initiation is altered and, concomitantly, the transcription complex formed on the mutant template remains unstable. These results suggest that there is a distinct topological requirement for complex formation which may involve an exact stereospecific alignment of RNA polymerase II with transcription factors bound to the promoter. Despite the apparent involvement of RNA polymerase, transcription per se is not required for complex stability.

SWI5 instability may be necessary but is not sufficient for asymmetric HO expression in yeast.

Homothallic haploid yeast cells divide to produce a mother cell that switches mating type and a daughter cell that does not. This pattern is the result of HO endonuclease transcription exclusively in mother cells, and there only transiently in late G1 as cells undergo Start. SWI5 encodes an HO transcription factor that is expressed during the S, G2, and M phases of the cell cycle. The lack of synthesis of SWI5 during G1 is essential to prevent HO transcription in daughter cells. Thus, HO must be activated by SWI5 protein synthesized in the previous cell cycle if it is to be properly regulated. SWI5 is inherited by both mother and daughter cells, and we show here that most of it is rapidly degraded during early G1. More stable mutant SWI5 proteins cause daughter cells to switch mating type, suggesting that SWI5 destruction is necessary to prevent HO expression in daughters. We show further that mother cells can still express HO when stimulated to undergo Start after arrest in early G1 for several hours. We propose that a small fraction of the SWI5 protein inherited by mother cells is extremely stable and that the crucial difference between mothers and daughters with regard to HO transcription is their differential ability to sequester SWI5 in a stable form, possibly as a component of transcription complexes on the HO promoter.

Functional characterization of X. laevis U5 snRNA genes.

Xenopus laevis U5 snRNA genes are found in several genomic arrangements, represented by a predominant tandem repeat of 583 bp and other minor repeats. Several copies of the major tandem repeat have been cloned and expressed in Xenopus oocytes. The transcripts assemble into U5 snRNPs which are recognized by anti-Sm antibodies. We have identified functional elements in the U5 gene promoter. Although similar in organization to other U snRNA gene promoters, U5 contains significant differences and is more efficiently expressed than the Xenopus U2 gene in oocytes. The proximal sequence element (PSE), although homologous to a mammalian consensus for this region (Skuzeski et al., 1984), does not resemble the previously characterized Xenopus U1 and U2 PSEs closely in sequence. The ATGCAAAT (octamer) part of the distal sequence element (DSE 1) is found in U5 in the orientation opposite to that in U1 and U2 gene promoters. DNase I protection experiments led to the identification of a third element (DSE 2), situated close to the octamer motif. Analysis of deletion mutants showed that both DSE 1 and 2 are essential parts of the U5 gene enhancer, and provides evidence that U snRNA enhancers are complex structures consisting of more than one site of DNA-factor interaction.

Only two of the four sites of interaction with nuclear factors within the Xenopus U2 gene promoter are necessary for efficient transcription.

An analysis, performed by DNase I footprinting, of the interactions between factors present in Molt-4 nuclear extracts and a Xenopus U2 snRNA gene promoter is presented. Four distinct regions of sequence-specific DNA-factor interaction are found. Two of these correspond to the previously identified proximal and distal sequence elements (PSE and DSE) of the promoter. Both of these elements are important in U2 transcription, indicating a functional role for the observed interactions. The other two sites of interaction correspond to a sequence element conserved in many, but not all, vertebrate U snRNA gene promoters (the MSE) and to a region adjacent to the site of transcription initiation (the "cap site"). Site-directed mutants of these latter two elements are constructed which no longer bind nuclear factors. Transcriptional analysis in Xenopus oocytes reveals that these mutants are transcribed as efficiently as wild-type U2. Other possible roles for the two factors are discussed.

The Xenopus U2 gene PSE is a single, compact, element required for transcription initiation and 3' end formation.

The proximal sequence element (PSE) of a Xenopus U2 snRNA gene has been analysed by extensive local mutagenesis. The PSE is compact, lying between -61 and -50 bp upstream of the transcription start site and is involved in signalling both transcription initiation and 3' end formation. No PSE mutants were found in which these two activities were differentially affected. Analysis of U2 gene promoters mutant in both the PSE and DSE failed to reveal any evidence for multiple signals involved in 3' end formation, leading to the conclusion that the PSE is the only promoter element required for this function. The U2 and U6 PSEs, which direct either pol II or both pol II and pol III transcription respectively, are shown to be functionally interchangeable. Apparent differences in human and Xenopus U2 gene PSE structure are discussed.

Changes in a SWI4,6-DNA-binding complex occur at the time of HO gene activation in yeast.

The yeast HO gene is transcribed transiently during G1 as cells undergo START. START-specific HO activation requires two proteins, SWI4 and SWI6, which act via a motif (CACGA4) repeated up to 10 times within the URS2 region of the HO promoter. We identified a DNA-binding activity containing SWI4 and SWI6 that recognizes the CACGA4 sequences within URS2. Two forms of SWI4,6-DNA complexes called L and U can be distinguished by their electrophoretic mobility. L complexes can be detected at all stages of the cell cycle, but U complexes are only detected in cells that have undergone START. The formation of U complexes may be the trigger of HO activation. The SWI6 protein is concentrated in the nucleus throughout G1, but at some point in S or G2 significant amounts accumulate in the cytoplasm. This change in cellular location of the SWI6 protein might contribute to the turnoff of HO transcription after cells have undergone START.

A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II.

The structure of a Xenopus U6 gene promoter has been investigated. Three regions in the 5'-flanking sequences of the gene that are important for U6 expression are defined. Deletion of the first, between positions -156 and -280 relative to the site of transcription initiation, reduces transcription to roughly 5% of its original level. Deletion of the second, between -60 and -77, abolishes transcription. These regions contain not only functional but also sequence homology to the previously defined distal and proximal sequence elements (DSE and PSE) of the Xenopus U2 promoter, although U2 is transcribed by RNA polymerase II and U6 by RNA polymerase III. Competition experiments show that at least the distal sequence elements of the two promoters bind to a common factor both in vivo and in vitro. Part of the sequence recognized by this factor is the octamer motif (ATG-CAAAT). A sequence similar to the common RNA polymerase II TATA box is also shown to have an effect, albeit minor, on U6 transcription. The U6 coding region contains a good match to the A box, part of all previously characterized RNA polymerase III promoters. Deletion of this region has no apparent effect on the efficiency or accuracy of U6 transcription.

Properties of a U1 RNA enhancer-like sequence.

The properties of a X.laevis U1B snRNA gene enhancer have been studied by microinjection in Xenopus oocytes. The enhancer-like sequence, defined as a short DNA stretch that is able to activate transcription in an orientation independent manner, is interchangeable between different U snRNA genes. The enhancer sequence alone does not, however, efficiently activate transcription from an SV40 pol II promoter but regains its activity when combined with the U-gene specific proximal sequence element. DNase I protection experiments show that the X.laevis U1B enhancer can interact specifically with a nuclear factor present in mammalian cells.

A transcription factor which binds to the enhancers of SV40, immunoglobulin heavy chain and U2 snRNA genes.

In eukaryotes the transcriptional control of RNA polymerase II-mediated gene expression is exerted by cis-acting regulatory DNA elements classified as promoter and enhancer sequences. These elements are composed of a number of different protein binding sites. The regulatory factors that recognize such 'modules' may be ubiquitous, tissue- or stage-specific, and positively or negatively acting. According to this model the transcriptional activity of a given gene is programmed by a combination of different modules. We analysed such a site of protein-DNA interaction, the octamer motif, in the enhancers of the simian virus (SV40) early genes and the murine immunoglobulin heavy-chain gene, and in the distal sequence element (DSE) of the U2 small nuclear (sn)RNA gene of Xenopus laevis. The corresponding DNA-binding factor appears to be the same in the three cases. Moreover, a fraction containing partially purified octamer motif binding factor has a stimulatory effect on transcription in an in vitro system.

The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5.

The intracellular localization of the S. cerevisiae transcription factor SWI5 is cell cycle dependent. The protein is nuclear in G1 cells but cytoplasmic in S, G2, and M phase cells. We have identified SWI5's nuclear localization signal (NLS) and show that it can confer cell cycle-dependent nuclear entry to a heterologous protein. Located within or close to the NLS are three serine residues, mutation of which results in constitutive nuclear entry. These residues are phosphorylated in a cell cycle-dependent manner in vivo, being phosphorylated when SWI5 is in the cytoplasm and dephosphorylated when it is in the nucleus. As all three serines are phosphorylated by purified CDC28-dependent H1 kinase activity in vitro, we propose a model in which the CDC28 kinase acts directly to control nuclear entry of SWI5.