Characterization of the Transcriptional Activity of the Cryptic Plasmid pRN1 from Sulfolobus islandicus REN1H1 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.

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Genetic dissection of centromere function.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3156 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3156-3166 ◽

Cited By ~ 10

Author(s):

I G Schulman ◽

K Bloom

Keyword(s):

Chromosome Segregation ◽

Copy Number ◽

Artificial Chromosome ◽

Plasmid Copy Number ◽

Genetic Dissection ◽

Negative Effects ◽

Plasmid Copy ◽

Complex Cells ◽

Copy Number Control

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.

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An unusually long-lived antisense RNA in plasmid copy number control: in vivo RNAs encoded by the streptococcal plasmid pIP501

Journal of Molecular Biology ◽

10.1006/jmbi.1996.0023 ◽

1996 ◽

Vol 255 (2) ◽

pp. 275-288 ◽

Cited By ~ 27

Author(s):

Sabine Brantl ◽

E. Gerhart H. Wagner

Keyword(s):

Antisense Rna ◽

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Copy Number Control

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Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194.

Journal of Bacteriology ◽

10.1128/jb.137.1.635-643.1979 ◽

1979 ◽

Vol 137 (1) ◽

pp. 635-643 ◽

Cited By ~ 101

Author(s):

B Weisblum ◽

M Y Graham ◽

T Gryczan ◽

D Dubnau

Keyword(s):

Copy Number ◽

Plasmid Copy Number ◽

High Copy Number ◽

Plasmid Copy ◽

Copy Number Control ◽

Isolation And Characterization

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Genetic dissection of centromere function

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3156-3166.1993 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3156-3166

Author(s):

I G Schulman ◽

K Bloom

Keyword(s):

Chromosome Segregation ◽

Copy Number ◽

Artificial Chromosome ◽

Plasmid Copy Number ◽

Genetic Dissection ◽

Negative Effects ◽

Plasmid Copy ◽

Complex Cells ◽

Copy Number Control

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The replication protein of the Sulfolobus islandicus plasmid pRN1

Biochemical Society Transactions ◽

10.1042/bst0320240 ◽

2004 ◽

Vol 32 (2) ◽

pp. 240-244 ◽

Cited By ~ 19

Author(s):

G. Lipps

Keyword(s):

Dna Polymerase ◽

Genome Sequence ◽

Biochemical Characterization ◽

Replication Protein ◽

Gene Products ◽

Genetic Element ◽

Sulfolobus Islandicus ◽

Basic Characteristics

The thermoacidophile crenarchaeote Sulfolobus ssp. is one of the best-studied Archaea. Cryptic and conjugative plasmids as well as viruses have been described for this genus. For the majority of the genetic elements only the genome sequence and the basic characteristics were determined. In contrast the fusellovirus SSV1 and the cryptic plasmid pRN1, which is the smallest known genetic element of the crenarchaeota, have been studied in more detail. The three gene products of the plasmid pRN1 have been characterized biochemically. The replication protein of the plasmid, a multifunctional enzyme, has a novel domain, termed prim/pol domain. This domain constitutes the first member of the DNA polymerase family E. Based on the biochemical characterization of the gene products a model of how pRN1 is replicated in vivo is proposed.

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Transcriptional Analysis of the Genetic Element pSSVx: Differential and Temporal Regulation of Gene Expression Reveals Correlation between Transcription and Replication

Journal of Bacteriology ◽

10.1128/jb.00638-07 ◽

2007 ◽

Vol 189 (17) ◽

pp. 6339-6350 ◽

Cited By ~ 18

Author(s):

Patrizia Contursi ◽

Raffaele Cannio ◽

Santina Prato ◽

Qunxin She ◽

Mosè Rossi ◽

...

Keyword(s):

Copy Number ◽

Regulation Of Gene Expression ◽

Growth Cycle ◽

Plasmid Copy Number ◽

Transcriptional Analysis ◽

Transcriptional Unit ◽

Plasmid Copy ◽

Temporal Regulation ◽

Sulfolobus Islandicus ◽

Transcription And Replication

ABSTRACT pSSVx from Sulfolobus islandicus strain REY15/4 is a hybrid between a plasmid and a fusellovirus. A systematic study performed by a combination of Northern blot analysis, primer extension, and reverse transcriptase PCR revealed the presence of nine major transcripts whose expression was differentially and temporally regulated over the growth cycle of S. islandicus. The map positions of the RNAs as well as the clockwise and the anticlockwise directions of their transcription were determined. Some genes were clustered and appeared to be transcribed as polycistronic messengers, among which one long transcriptional unit comprised the genes for the plasmid copy number control protein ORF60 (CopG), ORF91, and the replication protein ORF892 (RepA). We propose that a termination readthrough mechanism might be responsible for the formation of more than one RNA species from a single 5′ end and therefore that the nine different RNAs corresponded to only seven different transcriptional starts. Three transcripts, ORF76 and two antisense RNAs, countertranscribed RNA1 (ctRNA1) and ctRNA2, were found to be specifically expressed during (and hence correlated to) the phase in which the pSSVx copy number is kept under stringent control, as they were completely switched off upon the onset of the induction of replication.

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Biochemical characterization of Yin Yang 1 – RNA complexes

Biochemistry and Cell Biology ◽

10.1139/o07-155 ◽

2008 ◽

Vol 86 (1) ◽

pp. 31-36 ◽

Cited By ~ 12

Author(s):

Zachery R. Belak ◽

Andrew Ficzycz ◽

Nick Ovsenek

Keyword(s):

Rna Binding ◽

Biochemical Characterization ◽

Ribonucleoprotein Complexes ◽

Yin Yang 1 ◽

Significant Difference ◽

Yin Yang ◽

Rna Interaction ◽

Rna Complexes

YY1 (Yin Yang 1) is present in the Xenopus oocyte cytoplasm as a constituent of messenger ribonucleoprotein complexes (mRNPs). Association of YY1 with mRNPs requires direct RNA-binding activity. Previously, we have shown YY1 has a high affinity for U-rich RNA; however, potential interactions with plausible in vivo targets have not been investigated. Here we report a biochemical characterization of the YY1–RNA interaction including an investigation of the stability, potential 5′-methylguanosine affinity, and specificity for target RNAs. The formation of YY1–RNA complexes in vitro was highly resistant to thermal, ionic, and detergent disruption. The endogenous oocyte YY1–mRNA interactions were also found to be highly stable. Specific YY1–RNA interactions were observed with selected mRNA and 5S RNA probes. The affinity of YY1 for these substrates was within an order of magnitude of that for its cognate DNA element. Experiments aimed at determining the potential role of the 7-methylguanosine cap on RNA-binding reveal no significant difference in the affinity of YY1 for capped or uncapped mRNA. Taken together, the results show that the YY1–RNA interaction is highly stable, and that YY1 possesses the ability to interact with structurally divergent RNA substrates. These data are the first to specifically document the interaction between YY1 and potential in vivo targets.

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