Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system

Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. The enzyme contains 10 phage-like conserved motifs, two pentatricopeptide motifs and a serine-rich stretch. The protein expressed in insect cells supports transcription elongation in a promoter-independent assay. Addition of recombinant mtDBP caused arrest of the transcribing mtRNAP when the enzyme approached the mtDBP-binding site in the direction of transcription of mtDNA l-strand. When the polymerase encountered the protein-binding site in the opposite direction, termination occurred in a protein-independent manner, inside the mtDBP-binding site. Pulse-chase experiments show that mtDBP caused true transcription termination rather than pausing. These data indicate that mtDBP acts as polar termination factor and suggest that transcription termination in sea urchin mitochondria could take place by two alternative modes based on protein-mediated or sequence-dependent mechanisms

Direct evidence for phosphorus limitation on Amazon forest productivity

The productivity of rainforests growing on highly weathered tropical soils is expected to be limited by phosphorus availability1. Yet, controlled fertilization experiments have been unable to demonstrate a dominant role for phosphorus in controlling tropical forest net primary productivity. Recent syntheses have demonstrated that responses to nitrogen addition are as large as to phosphorus2, and adaptations to low phosphorus availability appear to enable net primary productivity to be maintained across major soil phosphorus gradients3. Thus, the extent to which phosphorus availability limits tropical forest productivity is highly uncertain. The majority of the Amazonia, however, is characterized by soils that are more depleted in phosphorus than those in which most tropical fertilization experiments have taken place2. Thus, we established a phosphorus, nitrogen and base cation addition experiment in an old growth Amazon rainforest, with a low soil phosphorus content that is representative of approximately 60% of the Amazon basin. Here we show that net primary productivity increased exclusively with phosphorus addition. After 2 years, strong responses were observed in fine root (+29%) and canopy productivity (+19%), but not stem growth. The direct evidence of phosphorus limitation of net primary productivity suggests that phosphorus availability may restrict Amazon forest responses to CO2 fertilization4, with major implications for future carbon sequestration and forest resilience to climate change.The authors acknowledge funding from the UK Natural Environment Research Council (NERC), grant number NE/L007223/1. This is publication 850 in the technical series of the BDFFP. C.A.Q. acknowledges the grants from Brazilian National Council for Scientific and Technological Development (CNPq) CNPq/LBA 68/2013, CNPq/MCTI/FNDCT no. 18/2021 and his productivity grant. C.A.Q., H.F.V.C., F.D.S., I.A., L.F.L., E.O.M. and S.G. acknowledge the AmazonFACE programme for financial support in cooperation with Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Institute of Amazonian Research as part of the grants CAPES-INPA/88887.154643/2017-00 and 88881.154644/2017-01. T.F.D. acknowledges funds from FundacAo de Amparo a Pesquisa do Estado de SAo Paulo (FAPESP), grant 2015/50488-5, and the Partnership for Enhanced Engagement in Research (PEER) programme grant AID-OAA-A-11-00012. L.E.O.C.A. thanks CNPq (314416/2020-0)

Cloning

of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination syste

Sea urchin mtDBP is a two-faced transcription termination factor with a biased polarity depending on the RNA polymerase

The sea urchin mitochondrial displacement (D)-loop binding protein mtDBP has been previously identified and cloned. The polypeptide (348 amino acids) displays a significant homology with the human mitochondrial transcription termination factor mTERF. This similarity, and the observation that the 3′ ends of mitochondrial RNAs coded by opposite strands mapped in correspondence of mtDBP-binding sites, suggested that mtDBP could function as transcription termination factor in sea urchin mitochondria. To investigate such a role we tested the capability of mtDBP bound to its target sequence in the main non-coding region to affect RNA elongation by mitochondrial and bacteriophage T3 and T7 RNA polymerases. We show that mtDBP was able to terminate transcription bidirectionally when initiated by human mitochondrial RNA polymerase but only unidirectionally when initiated by T3 or T7 RNA polymerases. Time-course experiments indicated that mtDBP promotes true transcription termination rather than transcription pausing. These results indicate that mtDBP is able to function as a bipolar transcription termination factor in sea urchin mitochondria. The functional significance of such an activity could be linked to the previously proposed dual role of the protein in modulating mitochondrial DNA transcription and replication

Purification of the sea urchin mtRNAP from baculovirus-infected insect cells and functional assay

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system"</p><p></p><p>Nucleic Acids Research 2007;35(7):2413-2427.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874651.</p><p>© 2007 The Author(s)</p> () Purification of mtRNAP by metal chelate affinity chromatography. The soluble portion of the insect cell lysate expressing the sea urchin mtRNAP was purified by Ni-NTA chromatography; cleared lysate, C.lys, flow-through, FT, wash, W, 3–5, fractions eluted at 250 mM imidazole, were separated on a 10% SDS–PAGE and revealed by immunoblotting as described in ‘Materials and Methods’ section. () Purification profile of mtRNAP as obtained by Heparin–Sepharose chromatography. Peak fractions from Ni-NTA column were pooled and subjected to Heparin–Sepharose chromatography. Input to the column (I) and fractions eluting between 0.75 and 0.9 M NaCl were analyzed by 7.5% SDS–PAGE and Coomassie Brilliant Blue stained. The molecular weight marker Precision Plus Protein Standards (Bio-Rad) is shown (M). The arrow inside the picture indicates the mtRNAP-containing band, as assessed by MALDI-TOF analysis. () Immunoblotting assay of input to the column (I) and Heparin–Sepharose eluted fractions. () Transcriptional activity of purified mtRNAP. The indicated Heparin–Sepharose fractions were assayed in the presence of [α-]PUTP, as described in ‘Materials and Methods’ section. On the top it is shown the diagram of the 71-bp tailed template, named 71bpDNA, with the open bar referring to the duplex DNA portion and the thin line to the 3′-tail. Run-off transcripts are indicated by arrowed line. Radiolabeled transcripts were separated on a 12% polyacrylamide/7M urea mini-gel followed by phosphorimaging analysis. 15 + R, fraction 15 treated with RNase A. RNA markers corresponding to the 10 nt ladder are indicated on the left