scholarly journals The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs.

1985 ◽  
Vol 100 (6) ◽  
pp. 1968-1976 ◽  
Author(s):  
N M Standart ◽  
S J Bray ◽  
E L George ◽  
T Hunt ◽  
J V Ruderman
Keyword(s):  
Sea Urchin ◽  
Ribonucleotide Reductase ◽  
Developmental Regulation ◽  
Large Subunit ◽  
Small Subunit ◽  
Affinity Column ◽  
Cdna Clones ◽  
Control Of Gene Expression ◽  
Alpha Tubulin ◽  
Sea Urchin Eggs

In both clam oocytes and sea urchin eggs, fertilization triggers the synthesis of a set of proteins specified by stored maternal mRNAs. One of the most abundant of these (p41) has a molecular weight of 41,000. This paper describes the identification of p41 as the small subunit of ribonucleotide reductase, the enzyme that provides the precursors necessary for DNA synthesis. This identification is based mainly on the amino acid sequence deduced from cDNA clones corresponding to p41, which shows homology with a gene in Herpes Simplex virus that is thought to encode the small subunit of viral ribonucleotide reductase. Comparison with the B2 (small) subunit of Escherichia coli ribonucleotide reductase also shows striking homology in certain conserved regions of the molecule. However, our attention was originally drawn to protein p41 because it was specifically retained by an affinity column bearing the monoclonal antibody YL 1/2, which reacts with alpha-tubulin (Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582). The finding that this antibody inhibits the activity of sea urchin embryo ribonucleotide reductase confirmed the identity of p41 as the small subunit. The unexpected binding of the small subunit of ribonucleotide reductase can be accounted for by its carboxy-terminal sequence, which matches the specificity requirements of YL 1/2 as determined by Wehland et al. (Wehland, J., H. C. Schroeder, and K. Weber, 1984, EMBO [Eur. Mol. Biol. Organ.] J., 3:1295-1300). Unlike the small subunit, there is no sign of synthesis of a corresponding large subunit of ribonucleotide reductase after fertilization. Since most enzymes of this type require two subunits for activity, we suspect that the unfertilized oocytes contain a stockpile of large subunits ready for combination with newly made small subunits. Thus, synthesis of the small subunit of ribonucleotide reductase represents a very clear example of the developmental regulation of enzyme activity by control of gene expression at the level of translation.

scholarly journals Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (ββ′) of Saccharomyces cerevisiae Ribonucleotide Reductase

2013 ◽  
Vol 288 (20) ◽  
pp. 13951-13959 ◽  
Author(s):  
Yan Zhang ◽  
Xiuxiang An ◽  
JoAnne Stubbe ◽  
Mingxia Huang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Small Subunit ◽  
Cluster Assembly ◽  
E Coli ◽  
Viability Assay ◽  
Docking Model

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.

scholarly journals Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA.

1986 ◽  
Vol 103 (6) ◽  
pp. 2129-2136 ◽  
Author(s):  
N Standart ◽  
T Hunt ◽  
J V Ruderman
Keyword(s):  
Enzyme Activity ◽  
Early Development ◽  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Large Subunit ◽  
Small Subunit ◽  
Maternal Mrna ◽  
Molecular Masses ◽  
Maternal Mrnas ◽  
Ribonucleotide Reductase Activity

Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase.

scholarly journals The Epstein-Barr Virus (EBV) Deubiquitinating Enzyme BPLF1 Reduces EBV Ribonucleotide Reductase Activity

2009 ◽  
Vol 83 (9) ◽  
pp. 4345-4353 ◽  
Author(s):  
Christopher B. Whitehurst ◽  
Shunbin Ning ◽  
Gretchen L. Bentz ◽  
Florent Dufour ◽  
Edward Gershburg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ribonucleotide Reductase ◽  
Active Site ◽  
Epstein Barr Virus ◽  
Reductase Activity ◽  
Large Subunit ◽  
Small Subunit ◽  
Deubiquitinating Enzyme ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT A newly discovered virally encoded deubiquitinating enzyme (DUB) is strictly conserved across the Herpesviridae. Epstein-Barr virus (EBV) BPLF1 encodes a tegument protein (3,149 amino acids) that exhibits deubiquitinating (DUB) activity that is lost upon mutation of the active-site cysteine. However, targets for the herpesviral DUBs have remained elusive. To investigate a predicted interaction between EBV BPLF1 and EBV ribonucleotide reductase (RR), a functional clone of the first 246 N-terminal amino acids of BPLF1 (BPLF1 1-246) was constructed. Immunoprecipitation verified an interaction between the small subunit of the viral RR2 and BPLF1 proteins. In addition, the large subunit (RR1) of the RR appeared to be ubiquitinated both in vivo and in vitro; however, ubiquitinated forms of the small subunit, RR2, were not detected. Ubiquitination of RR1 requires the expression of both subunits of the RR complex. Furthermore, coexpression of RR1 and RR2 with BPLF1 1-246 abolishes ubiquitination of RR1. EBV RR1, RR2, and BPLF1 1-246 colocalized to the cytoplasm in HEK 293T cells. Finally, expression of enzymatically active BPLF1 1-246 decreased RR activity, whereas a nonfunctional active-site mutant (BPLF1 C61S) had no effect. These results indicate that the EBV deubiquitinating enzyme interacts with, deubiquitinates, and influences the activity of the EBV RR. This is the first verified protein target of the EBV deubiquitinating enzyme.

Mutually exclusive synthetic pathways for sea urchin mitochondrial rRNA and mRNA

1989 ◽  
Vol 9 (3) ◽  
pp. 1069-1082
Author(s):  
D J Elliott ◽  
H T Jacobs
Keyword(s):  
16S Rrna ◽  
Sea Urchin ◽  
Large Subunit ◽  
Blot Hybridization ◽  
Small Subunit ◽  
12S Rrna ◽  
Mitochondrial Rna ◽  
Coding Region ◽  
Alternative Processing ◽  
Nadh Dehydrogenase Subunit 2

The structure and abundance of mitochondrial transcripts in sea urchin embryos were investigated by a combination of RNA blot-hybridization, S1 mapping, and primer extension assays. Between the egg and blastula stages, the relative abundance of mitochondrial rRNAs declined slightly, while that of mitochondrial mRNAs increased up to 10-fold. Fine mapping of the termini of the rRNAs and of the adjacent transcripts indicated that, although they appeared to be butt-joined at their 5' ends to the upstream transcripts, tRNA-Phe 5' to the small subunit (12S) rRNA and NADH dehydrogenase subunit 2 mRNA 5' to the large subunit (16S) rRNA, respectively, their 3' ends were found to overlap the 5' ends of the downstream transcripts. 12S rRNA was found to extend 7 to 13 nucleotides into the sequence of tRNA-Glu; 16S rRNA was shown to terminate 3 to 5 nucleotides inside the coding region of cytochrome oxidase subunit 1 (COI) and 8 to 10 nucleotides from the mapped 5' end of COI mRNA. The rRNAs and the downstream transcripts must therefore be synthesized by distinct pathways, either by alternative processing of the same primary transcript(s) or by processing of different precursors. In either case, the events which select the ribosomal 3' ends preclude the production of functional transcripts of the downstream genes from the same precursor molecule. No developmental alterations in transcript structure were detected. We propose that mitochondrial RNA levels are regulated in early development by the selection of alternate and mutually exclusive RNA-processing pathways.

scholarly journals Mutually exclusive synthetic pathways for sea urchin mitochondrial rRNA and mRNA.

1989 ◽  
Vol 9 (3) ◽  
pp. 1069-1082 ◽  
Author(s):  
D J Elliott ◽  
H T Jacobs
Keyword(s):  
16S Rrna ◽  
Sea Urchin ◽  
Large Subunit ◽  
Blot Hybridization ◽  
Small Subunit ◽  
12S Rrna ◽  
Mitochondrial Rna ◽  
Coding Region ◽  
Alternative Processing ◽  
Nadh Dehydrogenase Subunit 2

The structure and abundance of mitochondrial transcripts in sea urchin embryos were investigated by a combination of RNA blot-hybridization, S1 mapping, and primer extension assays. Between the egg and blastula stages, the relative abundance of mitochondrial rRNAs declined slightly, while that of mitochondrial mRNAs increased up to 10-fold. Fine mapping of the termini of the rRNAs and of the adjacent transcripts indicated that, although they appeared to be butt-joined at their 5' ends to the upstream transcripts, tRNA-Phe 5' to the small subunit (12S) rRNA and NADH dehydrogenase subunit 2 mRNA 5' to the large subunit (16S) rRNA, respectively, their 3' ends were found to overlap the 5' ends of the downstream transcripts. 12S rRNA was found to extend 7 to 13 nucleotides into the sequence of tRNA-Glu; 16S rRNA was shown to terminate 3 to 5 nucleotides inside the coding region of cytochrome oxidase subunit 1 (COI) and 8 to 10 nucleotides from the mapped 5' end of COI mRNA. The rRNAs and the downstream transcripts must therefore be synthesized by distinct pathways, either by alternative processing of the same primary transcript(s) or by processing of different precursors. In either case, the events which select the ribosomal 3' ends preclude the production of functional transcripts of the downstream genes from the same precursor molecule. No developmental alterations in transcript structure were detected. We propose that mitochondrial RNA levels are regulated in early development by the selection of alternate and mutually exclusive RNA-processing pathways.

scholarly journals Clb6-Cdc28 Promotes Ribonucleotide Reductase Subcellular Redistribution during S Phase

2017 ◽  
Vol 38 (6) ◽  
Author(s):  
Xiaorong Wu ◽  
Xiuxiang An ◽  
Caiguo Zhang ◽  
Mingxia Huang
Keyword(s):  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Small Subunit ◽  
S Phase ◽  
Nuclear Retention ◽  
Content Type ◽  
Dntp Pool ◽  
Checkpoint Kinases ◽  
Enhanced Sensitivity

ABSTRACTA tightly controlled cellular deoxyribonucleotide (deoxynucleoside triphosphate [dNTP]) pool is critical for maintenance of genome integrity. One mode of dNTP pool regulation is through subcellular localization of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step of dNTP biosynthesis. InSaccharomyces cerevisiae, the RNR small subunit, Rnr2-Rnr4, is localized to the nucleus, whereas the large subunit, Rnr1, is cytoplasmic. As cells enter S phase or encounter DNA damage, Rnr2-Rnr4 relocalizes to the cytoplasm to form an active holoenzyme complex with Rnr1. Although the DNA damage-induced relocalization requires the checkpoint kinases Mec1-Rad53-Dun1, the S-phase-specific redistribution does not. Here, we report that the S-phase cyclin–cyclin-dependent kinase (CDK) complex Clb6-Cdc28 controls Rnr2-Rnr4 relocalization in S phase. Rnr2 contains a consensus CDK site and exhibits Clb6-dependent phosphorylation in S phase. Deletion ofCLB6or removal of the CDK site results in an increased association of Rnr2 with its nuclear anchor Wtm1, nuclear retention of Rnr2-Rnr4, and an enhanced sensitivity to the RNR inhibitor hydroxyurea. Thus, we propose that Rnr2-Rnr4 redistribution in S phase is triggered by Clb6-Cdc28-mediated phosphorylation of Rnr2, which disrupts the Rnr2-Wtm1 interaction and promotes the release of Rnr2-Rnr4 from the nucleus.

cDNA cloning and gene expression of carbonic anhydrase in Chlamydomonas reinhardtii

1991 ◽  
Vol 69 (5) ◽  
pp. 1088-1096 ◽  
Author(s):  
Hideya Fukuzawa ◽  
Sarami Ishida ◽  
Shigetoh Miyachi
Keyword(s):  
Carbonic Anhydrase ◽  
Chlamydomonas Reinhardtii ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Mrna Level ◽  
Nuclear Genome ◽  
Small Subunit ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Upstream Gene

cDNA and genes encoding periplasmic carbonic anhydrase (CA) polypeptides of Chlamydomonas reinhardtii have been isolated and characterized. Nucleotide sequence analysis of cDNA clones revealed that the large subunit (35 kDa or 36.5 kDa) and the small subunit (4 kDa) are cotranslated as a precursor polypeptide (41 626 Da) with a NH2-terminal hydrophobic signal peptide of 20 amino acids. The amino acid sequence of Chlamydomonas CA showed 20–22% identity with animal CA isozymes (CAI, CAII, CAIII, and CAVII). Three zinc-liganded histidine residues and those forming the hydrogen-bond network to zinc-bound solvent molecules were highly conserved. No significant sequence similarity was observed between Chlamydomonas CA and chloroplast CAs of spinach and pea. Two copies of structurally related CA genes (CAH1 and CAH2) were tandemly clustered in Chlamydomonas nuclear genome and regulated by external CO2 concentration in a reverse manner. The 5′ upstream gene CAH1 encodes the major periplasmic CA whose mRNA level is induced under low CO2 condition in light. Photosynthesis is absolutely required for the accumulation of the CAH1 mRNA. The 3′ downstream gene CAH2 is possibly a gene for another periplasmic CA isozyme, which is induced under high CO2 conditions. Light has an inhibitory effect on the accumulation of the CAH2 mRNA. Key words: photosynthesis, light regulation, zinc, CO2-concentrating mechanism, intracellular processing.

scholarly journals Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (10) ◽  
pp. 6105-6113 ◽  
Author(s):  
M Huang ◽  
S J Elledge
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Essential Gene ◽  
Large Subunit ◽  
Signal Transduction Pathway ◽  
Transcriptional Response ◽  
Small Subunit ◽  
Cell Extracts

Ribonucleotide reductase (RNR), which catalyzes the rate-limiting step for deoxyribonucleotide production required for DNA synthesis, is an alpha2beta2 tetramer consisting of two large and two small subunits. RNR2 encodes a small subunit and is essential for mitotic viability in Saccharomyces cerevisiae. We have cloned a second essential gene encoding a homologous small subunit, RNR4. RNR4 and RNR2 appear to have nonoverlapping functions and cannot substitute for each other even when overproduced. The lethality of RNR4 deletion mutations can be suppressed by overexpression of RNR1 and RNR3, two genes encoding the large subunit of the RNR enzyme, indicating genetic interactions among the RNR genes. RNR2 and RNR4 may be present in the same reductase complex in vivo, since they coimmunoprecipitate from cell extracts. Like the other RNR genes, RNR4 is inducible by DNA-damaging agents through the same signal transduction pathway involving MEC1, RAD53, and DUN1 kinase genes. Analysis of DNA damage inducibility of RNR2 and RNR4 revealed partial inducibility in dun1 mutants, indicating a DUN1-independent branch of the transcriptional response to DNA damage.

Antisense oligonucleotides targeting the large subunit (R1) of human ribonucleotide reductase synergistically increase the cytotoxicity of doxorubicin and paclitaxel to MCF-7 breast cancer cells

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14626-e14626
Author(s):  
X. Jiang ◽  
R. L. Elliott ◽  
J. F. Head
Keyword(s):  
Breast Cancer ◽  
Ribonucleotide Reductase ◽  
Antisense Oligonucleotides ◽  
Large Subunit ◽  
Cancer Cell Line ◽  
Small Subunit ◽  
Chemotherapeutic Agents ◽  
Essential Enzyme ◽  
Dose Dependent ◽  
Mcf 7

e14626 Ribonucleotide reductase (RR) is an essential enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides for use in DNA synthesis. Human RR consists of two subunits, a large subunit (R1) and small subunit (R2). RR provides an attractive target for anticancer therapy. In the present study, we synthesized phosphorothioated antisenses oligonucleotides (RR1AS1, RR1AS2, RR1AS3, RR1AS4 and RR1AS5) that target the R1 subunit of RR. We treated the human breast cancer cell line MCF-7 with antisenses, and doxorubicin or paclitaxel for 72 hours. Cell proliferation was measured by 3[H]- thymidine incorporation. The effects of the drug combinations were analyzed with Biosoft Calcusyn software. The levels of RR mRNA were measured by RT-PCR. We found two antisenses, RR1AS2 and RR1AS4, inhibited the proliferation of MCF-7 cells in a dose-dependent pattern (IC50s: 5.32+1.64μm and 2.57+2.72μm, respectively, 72 hours incubation). Also, RR1AS2 and RR1AS4 significantly suppressed the expression of RR1 mRNA. When MCF-7 cells were incubated in media with a mixture of antisense and doxorubicin or paclitaxel, both RR1AS2 and RR1AS4 synergistically increased the cytotoxicity of doxorubicin and paclitaxel. Calcusyn analysis showed that averaged combination index (CI) were 0.59+0.04, 0.66+0.22, 0.83+0.16 and 0.88+0.03, when MCF-7 cells were treated with the mixtures of RR1AS2 + doxorubicin, RR1AS2 + paclitaxel, RR1AS4 + doxorubicin and RR1AS4 + paclitaxel, respectively (CI<1 indicates synergism). These results suggest that the combination of RR antisenses and chemotherapeutic agents, such as doxorubicin or paclitaxel, may decrease both the dosages and side effects of both antisense oligonucleotides and chemotherapeutic agents (doxorubicin and paclitaxel) in cancer therapy. No significant financial relationships to disclose.

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