Characterization of the Transcriptional Activity of the Cryptic Plasmid pRN1 fromSulfolobus islandicusREN1H1 and Regulation of Its Replication Operon

Abstract

The plasmid pRN1 from Sulfolobus islandicus REN1H1 belongs to the crenarchaeal plasmid family pRN. The plasmids in this family encode three conserved proteins that participate in plasmid replication and copy number regulation, as suggested by biochemical characterization of the recombinant proteins. In order to deepen our understanding of the molecular biology of these plasmids, we investigated the transcriptional activity of the model plasmid pRN1. We detected five major transcripts present at about 2 to 15 copies per cell. One long transcriptional unit comprises the genes for the plasmid-copy-number control protein Orf56/CopG and the replication protein Orf904. A second transcript with a long 3-untranslated region codes for the DNA binding protein Orf80. For both transcripts, we identified countertranscripts which could play a regulatory role. The function of the fifth transcript is unclear. For the five transcripts, we determined the start site, the transcript end, the stability, and the abundance in different growth phases. Reporter gene experiments demonstrated that the copy number control protein Orf56 represses transcription of the orf56-orf904 cotranscript in vivo.The plasmid pRN1 (5,350 bp) has been isolated from Sulfolobus islandicus strain REN1H1 (47) and occurs natively together with plasmid pRN2 in its host strain but has been shown to replicate independently of pRN2 (36). It is a member of the pRN family of genetic elements, comprising pRN1, pRN2, pDL10, pHEN7, and pSSVx (3,22,23,25,34). The more recently described plasmids pTIK4, pTAU4, pORA1 (18), and pIT3 (35) also contain open reading frames with sequence similarity to open reading frames from the pRN family plasmids.

Analysis of the First Genome of a Hyperthermophilic Marine Virus-Like Particle, PAV1, Isolated fromPyrococcus abyssi

“…Therefore, we cannot rule out the possibilities that additional transcripts may exist and that the longest version of T5 transcript represents only a minor fraction of the mRNA produced. Classical Northern experiments are commonly used to estimate both the size and relative abundance of major transcripts but are not reliable to detect transcripts of low abundance (7). Indeed, initial experiments to detect PAV1 transcripts by Northern analysis gave very weak and nonreproducible hybridization signals (not shown) probably because of the very low level of PAV1 transcripts under the culture conditions tested.…”

Molecular biology of the pRN1 plasmid from Sulfolobus islandicus

“…ORF56 assembles as a tetramer on its target binding site [5]. In addition, reporter gene experiments suggested that ORF56 acts as a repressor [6]. Since the genes orf56 and orf904 are co-transcribed and the latter gene encodes the replication protein, this type of negative-feedback control could be part of the copy-control mechanism of the plasmid.…”

Genetic analyses in the hyperthermophilic archaeon Sulfolobus islandicus

“…As a result, ten genomes of S. islandicus strains have been sequenced, eight from the U.S. DOE (Department of Energy) Joint Genome Institute (http://www.jgi.doe.gov/genomeprojects/) and two from the joint efforts of Danish, Chinese and French laboratories. Furthermore, many genetic elements including diverse viruses and plasmids have been isolated from S. islandicus and characterized [6][7][8][9][10][11][12][13][14], providing useful tools for studying molecular biology with this model.…”