Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase (original) (raw)
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The enantioselectivities of the active and allosteric sites of mammalian ribonucleotide reductase
FEBS Journal, 2005
Ribonucleotide reductases (RRs, EC 1.17.4.1) form a family of allosterically regulated enzymes that catalyze the conversion of ribonucleotides to 2¢-deoxyribonucleotides and are essential for de novo DNA biosynthesis and repair, regulating other enzymes in the DNA synthesis pathway via control of the nucleotide pool [1]. Of the four known classes of RR (Ia, Ib, II and III) class Ia, which requires two different subunits R1 and R2 for activity and catalyzes the reduction of all four common NDPs, is the most widespread, comprising all eukaryotic RRs as well as some from eubacteria, bacteriophages and viruses. The R1 subunit contains the active site as well as allosteric sites. We have recently demonstrated that there are three such sites in murine R1 (mR1) (the specificity or s-site, the adenine or a-site, and the hexamerization or h-site) [2,3], leading to a complex pattern of regulation of enzymatic activity, the major features of which are summarized in Scheme 1, as follows: (a) ATP, dATP, dGTP, or dTTP binding to the s-site drives formation of R1 2 ; (b) ATP or dATP binding to the a-site drives formation of R1 4 , which exists in two conformations, R1 4a and R1 4b , with the latter predominating at equilibrium; (c) ATP binding to the rather low affinity (K d 1-4 mm) h-site, which occurs at physiologically significant concentrations, drives formation of R1 6-dATP does not bind to this site at physiologically significant concentrations; (d) the R2 2 complexes of R1 2 , R1 4a , and R1 6 are enzymatically active, whereas the R2 2 complex of mR1 4b has little, if any, activity; and (e) the substrate specificity of RR is determined by the ligand occupying the s-site: ATP and dATP stimulate the reduction of CDP and UDP, dTTP stimulates the
Experimental Cell Research, 1988
The genes for the Ml subunit of the enzyme ribonucleotide reductase have been mapped in the human and the murine species by use of two independently derived mouse cDNA clones. Southern blot analysis of rodent x human somatic cell hybrid DNAs confirmed the assignment of RRMl to the short arm of human chromosome 11. In situ hybridization to human metaphase chromosomes revealed a peak of silver grains over the distal third of band 11~15, a region corresponding to subbands p15.4+pl5.5. The mouse Rrml locus was assigned to chromosome 7, where it forms part of a conserved syntenic group of at least seven other genes assigned to human chromosome band 11~15. 0 1988 Academic press, inc.
Journal of Biological Chemistry, 1999
We have examined the promoter of rnrB, the gene encoding the small subunit of ribonucleotide reductase of Dictyostelium discoideum, using lacZ as a reporter gene. Deletion analysis showed that expression of this gene in vegetative cells involves an A/T-rich element, whereas its expression in prespore cells during development requires a region encompassing two G/C-rich elements, designated box A and box B. Removal of boxes A and B results in very low level of activity. When either box A or box B is deleted, prestalk cells adjacent to the prespore zone also express -galactosidase. The behavior of these cis-regulatory elements implies that the mechanism regulating the prespore-specific expression of rnrB is different from that regulating other known prespore genes.
Enzymatically Active Mammalian Ribonucleotide Reductase Exists Primarily as an α6β2 Octamer
Journal of Biological Chemistry, 2006
Ribonucleotide reductase synthesizes deoxyribonucleotides, which are essential building blocks for DNA synthesis. The mammalian ribonucleotide reductase is described as an ␣ 2  2 complex consisting of R1 (␣) and R2 () proteins. ATP stimulates and dATP inhibits enzyme activity by binding to an allosteric site called the activity site on the R1 protein. Despite the opposite effects by ATP and dATP on enzyme activity, both nucleotides induce formation of R1 oligomers. By using a new technique termed Gas-phase Electrophoretic-Mobility Macromolecule Analysis (GEMMA), we have found that the ATP/ dATP-induced R1 oligomers have a defined size (hexamers) and can interact with the R2 dimer to form an enzymatically active protein complex (␣ 6  2). The newly discovered ␣ 6  2 complex can either be in an active or an inhibited state depending on whether ATP or dATP is bound. Our results suggest that this protein complex is the major form of ribonucleotide reductase at physiological levels of R1-R2 protein and nucleotides.
Gene, 1980
A set of recombinant DNAs with inserts of mRNA sequences coding for various mouse ribosomal proteins (r-proteins) were isolated. The recombinants were selected from a library of cDNA clones representing the small (~<12S) poly(A)+mRNA of mouse L ceils, which is relatively enriched for the r-protein mRNAs. Selection consisted of testing the various recombinant plasmids for their ability to hybridize selectively with an mRNA that directs the synthesis of a recognizable r-protein in a cell-free translational system. Translation products were subjected to a preliminary screen by electrophoresis on one-dimensional Triton X-100/urea slab gels and then more definitively identified by the two-dimensional gel analyses conventionally used for ribosomal proteins. The set of recombinants represents the mRNAs specifying ribosomal proteins S16, L7, L13, L18, L19, L30, 1_32/33 and probably L10. The recombinant plasmids were used as probes to determine the size and abundance of the corresponding mRNAs and their presumptive nuclear precursors. In general, there is a good correlation between mRNA size and the size of the encoded proteins, each rp-mRNA having a relatively uniform proportion (29 + 5%) of non-coding sequence. The pattern of poly(Af nuclear components was very distinctive for each of the rp-mRNAs, indicating that the majority of rp-mRNAs are probably not derived from common polycistronic transcripts. The largest detectable nuclear components were about 3-to 7-fold larger than their respective mature mRNAs. The individual rp-mRNAs appear to constitute about 0.06 to 0.12% of the poly(AfmRNA in L cells and about 0.06 to 0.38% in MPC-11 myeloma cells; for L cells this corresponds to about 300-400 copies of each mRNA per cell. The content of large nuclear molecules containing rp-mRNA sequences is estimated to be very low, on the order of only a few molecules per cell. Preliminary Southern blot analysis of EcoRI restriction fragments of mouse embryo DNA revealed very complex patterns, suggesting that a family of homologous sequences for the individual rp-mRNAs may exist in the mouse genome.
Structure of the rRNA genes in the hamster sperm nucleus
Journal of Andrology
We have examined the structure of the major ribosomal RNA (rRNA) genes in the hamster sperm nucleus, using flucrescent in situ hybridization (FISH). The rRNA genes are present as tandemly repeated clusters located at the telomenc ends of the short arms of five pairs of acrocentric chromosomes in the Syrian golden hamster (as they are in humans). In somatic cells, these five chromosome pairs come together to form the nucleolus, the site of rRNA synthesis. The nucleolus remains intact through S phase of the cell cycle, breaking apart only during late G2 and mitosis when the chromosomes condense. Mammalian sperm nuclei are the final products of meiotic division and morphological differentiation that includes a dramatic chromatin condensation. Consequently, it was not immediately obvious whether the rRNA genes would be condensed into a nucleolus-like structure in the mature spermatozoa, or separated, as they are In mitotic chromosomes. We found that of 117 sperm nuclei examined, 91.5% contained between two and five FISH signals for the rANA gene clusters, and 64.0% contained four (29%) or five (35%) signals. In decondensed hamster sperm nuclei, the rRNA hybridized signals were separated into independent strands. These data collectively indicate that the chromosomes containing the rRNA genes are not bound together into a pre-nucleolar structure in fully condensed mammalian sperm nuclei.
Structure, function, and mechanism of ribonucleotide reductases
Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, 2004
Ribonucleotide reductase (RNR) is the enzyme responsible for the conversion of ribonucleotides to 2V-deoxyribonucleotides and thereby provides the precursors needed for both synthesis and repair of DNA. In the recent years, many new crystal structures have been obtained of the protein subunits of all three classes of RNR. This review will focus upon recent structural and spectroscopic studies, which have offered deeper insight to the mechanistic properties as well as evolutionary relationship and diversity among the different classes of RNR. Although the three different classes of RNR enzymes depend on different metal cofactors for the catalytic activity, all three classes have a conserved cysteine residue at the active site located on the tip of a protein loop in the centre of an a/h-barrel structural motif. This cysteine residue is believed to be converted into a thiyl radical that initiates the substrate turnover in all three classes of RNR. The functional and structural similarities suggest that the present-day RNRs have all evolved from a common ancestral reductase. Nevertheless, the different cofactors found in the three classes of RNR make the RNR proteins into interesting model systems for quite diverse protein families, such as diiron-oxygen proteins, cobalamin-dependent proteins, and SAM-dependent iron-sulfur proteins. There are also significant variations within each of the three classes of RNR. With new structures available of the R2 protein of class I RNR, we have made a comparison of the diiron centres in R2 from mouse and Escherichia coli. The R2 protein shows dynamic carboxylate, radical, and water shifts in different redox forms, and new radical forms are different from non-radical forms. In mouse R2, the binding of iron(II) or cobalt(II) to the four metal sites shows high cooperativity. A unique situation is found in RNR from baker's yeast, which is made up of heterodimers, in contrast to homodimers, which is the normal case for class I RNR. Since the reduction of ribonucleotides is the rate-limiting step of DNA synthesis, RNR is an important target for cell growth control, and the recent finding of a p53-induced isoform of the R2 protein in mammalian cells has increased the interest for the role of RNR during the different phases of the cell cycle.
Endocrinology, 1998
During spermatogenesis, the levels of cAMP in seminiferous tubules undergo stage-dependent cyclical fluctuations. We show that changes in cAMP levels are accompanied by alternative exon splicing of the RNA encoding the cAMP-responsive transcription factor CREB (cAMP response element-binding protein), expressed in both the Sertoli and germ cells. Exons Y and W are expressed exclusively in the testis, and they introduce stop codons into the normal protein coding frame of CREB. The splicing in of W was shown earlier to activate the internal translation of two alternative products of the CREB messenger RNA (mRNA) containing the DNA-binding domain (I-CREBs). The I-CREBs act as potent inhibitors of activator isoforms of CREB. The functions of the alternatively spliced exon Y are unknown. To investigate whether the splicing of exons W and Y is regulated during spermatogenesis, seminiferous tubules, isolated from adult rats, were dissected into segments representing different stages of the spermatogenic cycle and were analyzed by RT-PCR. The analyses of pooledtubule segments revealed stage-dependent splicing of both exons W and Y in the CREB transcripts. Single tubules were dissected into smaller segments for greater staging accuracy and were analyzed by RT-PCR for CREB mRNAs containing either exons W or Y, as well as for FSH receptor mRNA. This analysis confirmed that a marked, cycle-dependent variation in CREB mRNA levels was occurring. Maximal splicing of exons W and Y occurs independently at different stages of the spermatogenic cycle, stages II-VI and IX, respectively. The distinct spermatogenic cycle-dependent regulation of the splicing of exons W and Y provides further evidence in support of a functional relevance for CREB-W and Y mRNA isoforms in spermatogenesis. (Endocrinology 139: 3721-3729, 1998) Materials and Methods Oligonucleotides Primer pairs were designed to distinguish transcripts including or lacking exons Y and W. Primers were directed to exons flanking the alternatively spliced exons (see Fig. 1). For the detection of CREB-Y, the forward primer was based in exon C, in preference to the infrequently spliced exon D.
Biology of Reproduction, 1994
Spermatogenic and Sertoli cells isolated from the mouse synthesize different proportions of the two mannose 6-phosphate receptors (MPR) during overnight culture periods (O'Brien et al., Endocrinology 1989; 125:2973). To determine the relative expression of MPR mRNAs in these cells, poly(A) + RNAs were examined by Northern blot analysis using cDNA probes specific for the cation-independent (CI) and cation-dependent (CD) MPRs. A single CI-MPR transcript,-10 kb in size, was present in all tissues and cell types examined. Like the CI-MPR protein, this transcript was more abundant in Sertoli cells than in spermatogenic cells isolated from adult testes. The CD-MPR is the predominant MPR synthesized by pachytene spermatocytes or round spermatids. Multiple CD-MPR transcripts were detected in these cells, including a 2.4-kb CD-MPR mRNA that was indistinguishable from CD-MPR transcripts in somatic tissues and Sertoli cells. Smaller CD-MPR mRNAs of-1.4 and 1.6 kb were prominent in pachytene spermatocytes and round spermatids, respectively, but were faint or undetectable in somatic tissues. These smaller CD-MPR mRNAs did not hybridize with an 0.9-kb restriction fragment derived from the CD-MPR 3' untranslated region (UTR), suggesting that alternate polyadenylation signals are used to produce multiple CD-MPR transcripts in spermatogenic cells. When poly(A) tracts were selectively removed from germ cell RNAs by ribonuclease H treatment, identical 1.3-kb CD-MPR mRNAs were detected in pachytene spermatocytes and round spermatids, indicating that the size difference between the 1.4and 1.6kb transcripts is due to variations in poly(A) tail length. These alterations in the 3' UTR of the CD-MPR transcripts may affect mRNA stability or translation during spermatogenesis.