Ribonucleotide Reductase R2 Research Articles

Defining a Novel cis-Element in the 3′-Untranslated Region of Mammalian Ribonucleotide Reductase Component R2 mRNA: cis-trans-INTERACTIONS AND MESSAGE STABILITY

Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The 3'-untranslated region (3'-UTR) of mammalian ribonucleotide reductase R2 mRNA has been implicated in the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate-mediated stabilization of mouse BALB/c 3T3 R2 message. We investigated the possibility that the 3'-UTR contains regulatory information for R2 mRNA turnover. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 9-nucleotide cis-element, 5'-UCGUGUGCU-3', which interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) in a sequence-specific manner to form a 45-kDa R2 binding protein complex. The binding activity was redox-sensitive and down-regulated by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid in a dose-dependent manner. Insertion of a 154-base pair fragment containing the cis-element led to markedly reduced accumulation of chloramphenicol acetyltransferase hybrid mRNA relative to the same insert carrying a series of G --> A mutations within this element that eliminated binding. We suggest that the 9-nucleotide region functions as a destabilizing element. These results provide a model for ribonucleotide reductase gene expression through a novel and specific mRNA cis-trans-interaction involving a phosphorylation signal pathway that leads to changes in the stability of R2 message.

Modeling the Oxygen Activation Chemistry of Methane Monooxygenase and Ribonucleotide Reductase

The authors review two different aspects of their work concerned with modeling enzymes responsible for the activation of dioxygen: (i) design of a non-heme diiron-O{sub 2} adduct, and (ii) synthesis of high-valent bis({mu}-oxo)diiron reaction intermediates. The authors propose structures for intermediates in the methane monooxygenase and the ribonucleotide reductase R2 reaction cycles. 53 refs., 9 figs.

A link between ferritin gene expression and ribonucleotide reductase R2 protein, as demonstrated by retroviral vector mediated stable expression

We have constructed a retroviral expression vector for the mammalian ribonucleotide reductase R2 component. Stable infectants, which express a myc epitope tagged R2 protein from the vector cDNA were obtained and described for the first time. Cells containing the recombinant protein exhibited increased ribonucleotide reductase activity, and were resistant to the antitumour agent hydroxyurea, which targets the R2 component of ribonucleotide reductase. Furthermore, a direct link between ferritin gene expression and R2 protein was observed, since cells containing vector expressed recombinant R2 protein exhibited increased H-chain and L-chain ferritin gene expression.

The Ribonucleotide Reductase R1 Inhibitor N-Acetyl-N,O-di(propylcarbamoyl)hydroxylamine, an Analogue of Caracemide

The molecular structure of the caracemide analogue N-acetyl-N,O-di(propylcarbamoyl)hydroxylamine (Chemical Abstracts nomenclature N-[(propylamino)carbonyl]-N-{[(propylamino)carbonyl]oxy}acetamide), C10H19N3O4, is comparable to the structure of the parent compound N-acetyl-N,O-di(methylcarbamoyl)hydroxylamine. The caracemide moiety of the compound consists of two nearly planar moieties, which are almost perpendicular to one another as in the crystal structure of caracemide itself. The two propyl groups in each of the two molecules (A and B) in the asymmetric unit have different conformations. One of these groups adopts the gauche conformation, with torsion angles of 49.1 (6) and −61.3 (4)° for molecules A and B, respectively, while the other adopts a fully extended conformation, with respective torsion angles of 179.2 (3) and 176.5 (3)°. The main differences in bond lengths, angles and torsion angles between molecules A and B are found in one of the propyl groups.

Reversible Red-Ox Reactions of the Diiron Site in the Mouse Ribonucleotide Reductase R2 Protein

The red-ox reactions of the dinuclear iron center of mouse R2 protein upon interaction with different reductants (dithionite alone and with mediators) and oxidants (PES, methylene blue, hydrogen peroxide and para-benzoquinone) have been studied by EPR and optical spectroscopy. The obtained results indicate that the transitions between Fe(III)Fe(III), Fe(II)Fe(III), and Fe(II)Fe(II) states of the dinuclear iron center are reversible and the μ-oxo-bridge may be formed upon oxidation by non-oxygen oxidants. In contrast to the case for theE. coliR2 protein, dithionite alone reduces the tyrosyl radical and diiron center in mouse R2 protein.

The TATA-less Promoter of Mouse Ribonucleotide Reductase R1 Gene Contains a TFII-I Binding Initiator Element Essential for Cell Cycle-regulated Transcription

Mammalian ribonucleotide reductase shows S-phase specific expression and consists of two non-identical subunits, proteins R1 (large subunit) and R2 (small subunit). A comparison between the human and mouse TATA-less R1 gene promoters revealed four highly conserved DNA regions, while the remaining sequence showed a low degree of conservation. Two regions, alpha and beta, were earlier identified as protein binding regions in the mouse R1 promoter by using DNase footprinting technique. The two new regions are located to the transcription start and to a DNA sequence about 40 base pairs downstream from the start. Gel shift assays using TFII-I antibodies and competition with an oligonucleotide representing the terminal deoxynucleotidyl transferase inhibitor element identified the start region as a TFII-I binding initiator element. The conserved downstream region, called gamma, also formed specific DNA-protein complexes in gel shift assays. Functional studies, using synchronized cells stably transformed by R1 promoter-luciferase reporter gene constructs, indicated that the initiator and the gamma elements together were necessary for cell cycle-regulated R1 promoter activity. Earlier published data, indicating Sp1 binding to the R1 alpha/beta regions, could not be confirmed, suggesting that the R1 initiator element may function independent of Sp1.

Role of a Proximal NF-Y Binding Promoter Element in S Phase-specific Expression of Mouse Ribonucleotide Reductase R2 Gene

Cell cycle-regulated transcription of the R2 gene of mouse ribonucleotide reductase was earlier shown to be controlled at the level of elongation by an S phase-specific release from a transcriptional block. However, the R2 promoter is activated very early when quiescent cells start to proliferate, and this activation is dependent on three upstream sequences located nucleotide -672 to nucleotide -527 from the transcription start. In this study, we use R2-luciferase reporter gene constructs and gel shift assays to demonstrate that, in addition to the upstream sequences, a proximal CCAAT element specifically binding the transcription factor NF-Y is required for continuous activity of the R2 promoter through the S phase. When the CCAAT element is deleted or mutated, promoter activity induced by the upstream elements decays before cells enter S phase, and the transcriptional block is released. This is a clear example of how changing of a proximal sequence element can alter not only the quantitative but also the qualitative response to upstream transcription activation domains.

Improved Adenovirus Vector Provides Herpes Simplex Virus Ribonucleotide Reductase R1 and R2 Subunits Very Efficiently

We have constructed a new adenovirus (Ad) expression vector, pAdBM5, that allows for the production of unprecedented levels of recombinant protein in the human 293 cell line using the Ad expression system. The main feature of this vector is a combination of enhancer sequences that increases the activity of the ectopic major late promoter (MLP) in recombinant Ad. In 293 cells infected with helper-free Ad recombinants generated with the pAdBM5 transfer vector, both herpes simplex virus (HSV) ribonucleotide reductase R1 and R2 subunits represent the most abundant polypeptides, accounting for as much as 15-20% of total cellular proteins. Our data suggest that this level of expression is probably very close to the upper limit of the system. Furthermore, when compared to the widely utilized baculovirus (Bac)/Sf9 expression system, the improved Ad vector showed a better performance for the production and purification of active HSV-2 ribonucleotide reductase R1 and R2 subunits. The R2 subunit was about 5-fold more abundant in recombinant Ad-infected 293 cells than in Bac-infected Sf9 cells while the R1 subunit was produced at roughly similar levels with either system. However, the amount of active soluble R1 obtained from recombinant Ad-infected 293 cells was at least 5 times higher because most of the R1 produced in Sf9 cells was insoluble.

Recombinant Desulfovibrio vulgaris rubrerythrin. Isolation and characterization of the diiron domain.

The gene encoding Desulfovibrio (D.) vulgaris rubrerythrin (Prickril, B. C., Kurtz, D. M., Jr., LeGall, J., & Voordouw, G. (1991) Biochemistry 30, 1118), a protein of unknown function containing both FeS4 and (mu-oxo)diiron sites, was cloned and overexpressed in Escherichia coli. Upon cell lysis, the overexpressed protein was found in an insoluble form deficient in iron. Iron was incorporated in vitro by dissolving the protein in 3 M guanidinium chloride, adding Fe(II) anaerobically and diluting the denaturant. This recombinant rubrerythrin was found to have properties very similar to those of rubrerythrin isolated from D. vulgaris, except that the recombinant rubrerythrin contained six rather than four (or five) iron atoms per 44 kDa homodimer. Analyses of UV-vis, Mössbauer, and EPR spectra showed that the six iron atoms in recombinant rubrerythrin are organized as two FeS4 and two (mu-oxo/hydroxo)diiron sites. In order to allow examination of the diiron sites in the absence of the FeS4 sites, a truncated gene encoding the N-terminal 152 residues of D. vulgaris rubrerythrin was also cloned and overexpressed as an insoluble protein in E. coli, and iron was incorporated by a procedure analogous to that for recombinant rubrerythrin. This so-called "chopped" rubrerythrin (CRr) was found to consist of an approximately 35 kDa homodimer containing four iron atoms. Spectroscopic characterization indicated that the four iron atoms in CRr are organized as two diiron sites, the majority of which closely resemble the (mu-oxo)diiron(III) sites in E. coli ribonucleotide reductase R2 protein, and a minor fraction of which resemble the mixed-valent diiron(II,III) site in methane monooxygenase hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Defining a novel cis element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA: role in transforming growth factor-beta 1 induced mRNA stabilization.

Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 1 induced message stability. Using end-labeled RNA fragments in gel shift assays and UV cross-linking analyses, we detected in the 3'-UTR a novel 9 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis element effectively competed out the formation of the R2 3'-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, we were able to predict the consensus sequences, 5'-GAGUUUNNN-3' and 5'-NNNUUUGAG-3' for optimal protein binding. These results support a model in which the 9 nt region functions in cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is involved in TGF-beta 1-mediated effects.

Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit.

Mammalian ribonucleotide reductase is a heterotetramer formed by the two non-identical homodimers proteins R1 and R2. We have succeeded in expressing the 90-kDa mouse R1 protein in Escherichia coli in an active, soluble form using the T7 RNA polymerase pET vector system. To avoid inclusion bodies, the bacteria were grown at 15 degrees C with minimal concentration of the inducer isopropyl-1-thio-beta-D-galactopyranoside. After a rapid purification procedure, approximately 20 mg of pure R1 protein were obtained per liter of bacterial culture. The concentrated R1 protein solution had a pinkish red color. Spectroscopy in combination with iron and labile sulfur analyses demonstrated that the color originated from an iron-sulfur complex. However, all attempts to demonstrate a function of this complex have been inconclusive. A comparison of the recombinant R1 protein with the corresponding protein purified from calf thymus showed no evidence for glycosylation. Circular dichroism spectroscopy indicated an alpha-helical content of 50%. A flexible COOH-terminal tail of 7 residues in the R2 protein was earlier shown to be essential for binding to the R1 protein. Using a peptide protection assay and photoaffinity labeling, we now show that the R2 protein tail interacts with a region close to the carboxyl terminus of the R1 protein.

Regulation of mammalian ribonucleotide reductase R1 mRNA stability is mediated by a ribonucleotide reductase R1 mRNA 3'-untranslated region cis-trans interaction through a protein kinase C-controlled pathway.

Ribonucleotide reductase catalyses the reaction that eventually provides the four deoxyribonucleotides required for the synthesis and repair of DNA. U.v.-cross-linking and band-shift experiments have identified in COS 7 monkey cells an approx. 57 kDa ribonucleotide reductase R1 mRNA-binding protein called R1BP, which binds specifically to a 49-nt region of the R1 mRNA 3'-untranslated region (3'UTR). The R1BP-RNA binding activity was down-regulated by the tumour promoters phorbol 12-myristate 13-acetate (PMA; 'TPA') and okadaic acid, and up-regulated by the protein kinase C inhibitor staurosporine, in a dose-dependent fashion. Furthermore, staurosporine treatment decreased the stability of R1 and CAT (chloramphenicol acetyltransferase)/R1 hybrid mRNAs, whereas PMA and okadaic acid increased the stability of these messages, in a dose-dependent manner. In contrast, treatment of cells with forskolin, a protein kinase A inhibitor, did not alter either R1BP-RNA binding or R1 mRNA-stability characteristics. Transfectants containing R1 or CAT/R1 cDNA constructs with a deletion of the 49-nt 3'UTR sequence failed to respond in message-stability studies to the effects of PMA, staurosporine or okadaic acid. These observations indicate that a protein kinase C signal pathway regulates ribonucleotide reductase R1 gene expression post-transcriptionally, through a mechanism involving a specific cis-trans interaction at a 49-nt region within the R1 mRNA 3'UTR.

Affinity of Synthetic Peptides for the HSV-2 Ribonucleotide Reductase R1 Subunit Measured with an Iodinated Photoaffinity Peptide

The herpes simplex virus (HSV) ribonucleotide reductase comprises two nonidentical subunits, R1 and R2, which associate to form the active holoenzyme. A sensitive binding assay was developed to measure the affinity of inhibitory peptides for the HSV R1 subunit. The assay involved the use of a photoreactive radioligand [4′-azido-Phe328,3′,5′-125I-Tyr329] HSV R2-(328-337), an analogue of the decapeptide Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu which corresponds to the C-terminal sequence (328-337) of the HSV R2 protein. As the radioligand binds covalently to the HSV R1 subunit upon uv irradiation, the affinity of peptide inhibitors can be easily determined by measuring their ability to compete with this highly specific binding. The method, which did not require any pure preparation of R1, was tested at 25 and 4°C and showed a significant increase in the affinity of the peptide inhibitors at 4°C. The relative affinity of these peptides was in agreement with their relative potency to inhibit reductase activity. The affinity of R2 subunit for R1 was also determined, and an IC50 of 0.05 μM was measured. Altogether, this assay represents a precise and reliable tool with which to study more potent HSV ribonucleotide reductase peptide inhibitors, and the method could be applied to the study of other protein-protein and peptide-protein interactions.

Phorbol ester modulation of a novel cytoplasmic protein binding activity at the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA and role in message stability.

The R2 gene of ribonucleotide reductase is elevated in BALB/c 3T3 fibroblasts treated with the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment increased the half-life of the R2 message by 3-fold, showing that TPA regulates R2 gene expression by a post-transcriptional mechanism(s). A 20-nucleotide (nt) TPA-responsive region was found within the R2 mRNA 3'-untranslated region (3'UTR). Ultraviolet cross-linking detected a novel 45-kDa protein-R2 mRNA complex migration band that bound selectively to the 20-nt fragment and did not bind to the 5'UTR or the coding region of the R2 message, or to the 3'UTRs of mRNA from several other genes, or to the homopolymer poly(A) sequence. The-45 kDa protein-R2 mRNA binding activity observed in unstimulated cells was markedly down-regulated after TPA treatment. Deletion of a 201-nt region, containing the 20-nt sequence, from the 3'UTR caused stabilization of hybrid chloramphenicol acetyltransferase mRNA in the absence of TPA treatment. Furthermore, in vitro decay reaction mixtures supplemented with the 20-nt sense RNA transcript resulted in stabilization of R2 message. A model is presented of R2 message regulation in which a cis-element within the 20-nt sequence of the 3'UTR interacts with a cytosolic protein to form a 45-kDa protein-mRNA binding complex. The TPA-induced alteration of R2 message stability is at least in part due to the down-regulation of the 45-kDa protein-mRNA binding activity which is linked to a reduction in the rate of R2 mRNA degradation.

Defining a novel ribonucleotide reductase r1 mRNA cis element that binds to an unique cytoplasmic trans-acting protein.

Ribonucleotide reductase is a highly regulated rate-limiting enzyme activity in DNA synthesis, responsible for reducing ribonucleotides to their deoxyribonucleotide forms. Using 3'-end labeled RNA and band-shift and UV cross-linking analyses, we have identified a cis-element, 5'-CAAACUUC-3', within the 3'-untranslated region of the mammalian ribonucleotide reductase R1 mRNA, which binds a cytoplasmic protein in BALB/c 3T3 mouse cells, to form a 57 kDa RNA-protein complex. Sequence-specific binding was observed, and binding was prevented by several different mutations within the cis-element. We suggest that this cis-trans interaction plays a role in R1 mRNA stability.

Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase.

Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins R1 and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase R1 gene was compiled from a number of overlapping lambda clones isolated from a Charon 4A mouse sperm genomic library. The R1-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the R1-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One complex most likely contained Sp1, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-dependent R1 gene expression.

Mammalian ribonucleotide reductase R1 mRNA stability under normal and phorbol ester stimulating conditions: involvement of a cis-trans interaction at the 3' untranslated region.

Ribonucleotide reductase R1 gene expression is elevated in BALB/c 3T3 fibroblasts treated with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). We show that TPA treatment increased the half-life of R1 mRNA by 5-fold, indicating that TPA regulates R1 gene expression by a post-transcriptional mechanism. We investigated the possibility that the 3' untranslated region (3'UTR) of R1 mRNA contains regulatory information for TPA-mediated message stability. Our studies demonstrated that a 49 nucleotide (nt) TPA-responsive region existed within the R1 mRNA 3'UTR. Deletion of the 49 nt region led to the abolishment of TPA-induced stability of R1 and hybrid CAT mRNAs. Further deletions of the 3'UTR did not significantly affect mRNA turnover rates. In addition, we detected by cross-linking a novel 52-57 kDa R1 mRNA-binding protein (R1BP) that bound selectively to the 49 nt region of the R1 mRNA 3'UTR and did not bind to the 5'UTR, the coding region or other mRNA 3'UTRs. The R1BP-RNA binding activity observed in unstimulated cells was rapidly and markedly down-regulated after TPA treatment, suggesting a role for R1BP in mRNA metabolism, and in the mechanism of action of TPA-induced R1 message stabilization. These results support a novel model of R1 gene regulation in which a cis-element(s) within the 49 nt region of the R1 mRNA 3'UTR interacts with R1BP in a mechanism that regulates R1 message stability. We propose that this model accounts for the TPA-mediated stability alteration of R1 message, through down-regulation of R1BP-RNA binding activity linked to a reduction in the rate of R1 mRNA degradation.

Complexes of a pendant phenoxyl radical ligand, including a new model for the ribonucleotide reductase R2 protein

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTComplexes of a pendant phenoxyl radical ligand, including a new model for the ribonucleotide reductase R2 proteinDavid P. Goldberg, Stephen P. Watton, Axel Masschelein, LeAnn Wimmer, and Stephen J. LippardCite this: J. Am. Chem. Soc. 1993, 115, 12, 5346–5347Publication Date (Print):June 1, 1993Publication History Published online1 May 2002Published inissue 1 June 1993https://doi.org/10.1021/ja00065a077RIGHTS & PERMISSIONSArticle Views178Altmetric-Citations18LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (291 KB) Get e-AlertsSupporting Info (2)»Supporting Information Supporting Information Get e-Alerts

A novel transforming growth factor-beta 1 responsive cytoplasmic trans-acting factor binds selectively to the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA: role in message stability.

Ribonucleotide reductase is a highly regulated enzyme that provides the four deoxyribonucleotides required for DNA synthesis. Our studies showed that TGF-beta 1 treatment of BALB/c 3T3 mouse fibroblasts markedly elevated ribonucleotide reductase R2 mRNA levels, and also increased the half-life of R2 message by 4-fold from 1.5 h in untreated cells to 6 h in treated cells. We describe a novel 75 Kd sequence-specific cytoplasmic factor (p75) that binds selectively to a 83-nucleotide 3'-untranslated region of R2 mRNA and did not bind to the 5'UTR, the coding region of the R2 message or to the 3'UTRs of other mRNAs (from c-myc, GM-CSF and the iron responsive element from the transferrin receptor mRNA), or to the homopolymer poly(A) sequence. p75-RNA binding activity, which requires new protein synthesis, is not present in untreated cells, but is induced following TGF-beta 1 stimulation. The in vivo kinetics of appearance of p75 binding activity paralleled the accumulation of R2 mRNA. Insertion of the 3'-untranslated region into the chloramphenicol acetyltransferase (CAT) message confers TGF-beta 1 induced stability of RNA in stably transfected cells, while the same insert carrying a deletion of the 83-nucleotide fragment had little affect on RNA levels. Furthermore, in vitro decay reactions that contained the 83-nucleotide RNA or deletion of this fragment caused a significant decrease in TGF-beta 1 stabilization of R2 message. A model is presented of R2 message regulation in which TGF-beta 1 mediated stabilization of R2 message involves a specific interaction of a p75-trans-acting factor with a cis-element(s) stability determinant within the 83-nucleotide sequence which is linked to a reduction in the rate of R2 mRNA degradation.

An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit.

Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression.