Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA

The RepA protein of plasmid R1 is rate-limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini-R1 derivatives and to lowered repA expression assayed in translational repA-lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA-mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication.     

A new moderately repetitive rat DNA sequence detected by a cloned 4.5 SI DNA

4.5 SI RNA is an abundant, noncapped, small nuclear RNA found in rodent cells. The 4.5 SI RNA is 98 or 99 nucleotides long and contains no modified nucleotides; it is synthesized by RNA polymerase III, is partly hydrogen-bonded to poly(A+) hnRNA, and was the first small nuclear RNA to be purified and sequenced (Busch, H., Reddy, R., Ruthblum, L., and Choi, Y. C. (1982) Annu. Rev. Biochem. 51, 617-654). In studies on the structure and organization of genes coding for this abundant RNA, it was found that this RNA is homologous to an apparently novel family of repetitive sequences. Two clones were characterized; one clone showed that its sequence is identical to the RNA in the first 92 residues and differed only in the last six nucleotides. In addition, the 3'-end of the sequence contained an A,T-rich region, and the sequence was flanked by a 15-nucleotide long direct repeat of AAAATATAGACACTG. The second clone characterized contained nucleotide sequences 1-57 corresponding to the RNA and was flanked by a 15-nucleotide long direct repeat. The structural features of these two DNAs are consistent with RNA-mediated DNA synthesis and integration of this DNA into the genome at random sites. It is estimated that there are about 10,000 copies of this family of sequences in the haploid rat genome.     

Identification and characterization of mutations responsible for a runaway replication phenotype of plasmid R1

Initiation of replication of the resistance plasmid R1 is carefully regulated by the two negatively acting factors, CopA and CopB. It is shown here that the temperature-dependent runaway-replication phenotype of an R1 plasmid mutant is caused by two point mutations in each of the promoters for the genes of these control factors. Expression of the two genes is affected in the following way: (1) one C-to-T transition in the putative -35 box of the copB-repA operon creates a two- to three-fold stronger promoter from which expression is temperature-dependent; (2) another C-to-T transition in a G + C-rich area immediately downstream from the -10 box of the copA promoter reduces expression of the copA gene three-fold. The phenotypic consequences of the two mutations are discussed in the light of the current model for R1 replication control.     

Replication control in plasmid R1: duplex formation between the antisense RNA, CopA, and its target, CopT, is not required for inhibition of RepA synthesis.

The replication frequency of plasmid R1 is regulated by an antisense RNA, CopA, which inhibits the synthesis of the rate-limiting initiator protein RepA. The inhibition requires an interaction between the antisense RNA and its target, CopT, in the leader of the RepA mRNA. This binding reaction has previously been studied in vitro, and the formation of a complete RNA duplex between the two RNAs has been demonstrated in vitro and in vivo. Here we investigate whether complete duplex formation is required for CopA-mediated inhibition in vivo. A mutated copA gene was constructed, encoding a truncated CopA which is impaired in its ability to form a complete CopA/CopT duplex, but which forms a primary binding intermediate (the 'kissing complex'). The mutated CopA species (S-CopA) mediated incompatibility against wild-type R1 plasmids and inhibited RepA-LacZ fusion protein synthesis. Northern blot, primer extension and S1 analyses indicated that S-CopA did not form a complete duplex with CopT in vivo since bands corresponding to RNase III cleavage products were missing. An in vitro analysis supported the same conclusion. These data suggest that formation of the 'kissing complex' suffices to inhibit RepA synthesis, and that complete CopA/CopT duplex formation is not required. The implications of these findings are discussed.     

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.     

Mini-exon derived RNA gene ofLeishmania donovani: structure,organization and expression

Mini-exon derived RNA is a small nuclear RNA of trypanosomatid protozoa such asLeishmania which donates its 5′-terminal 39 nucleotides to the 5’-ends of cellular messenger RNAs by trans-splicing. We have cloned a mini-exon derived RNA gene fromLeishmania donovani and studied its organization and expression. About 200 copies of the gene per haploid genome are organized as a tandem repeat on a single chromosome. The gene is transcribed as a 95-nucleotide RNA. The first 39 nucleotides of mini-exon derived RNA is also found at the 5′-terminus of a cellular mRNA (Β-tubulin), thus confirming its identity. Sequence analysis of the gene and its flanking regions showed that while classical RNA polymerase II promoter elements such as TATA and CAAT are absent from the 5′-upstream region, intragenic sequence motifs resembling RNA polymerase III promoter elements are present. The implications of this finding for mini-exon derived RNA expression are discussed.     

Structural and functional similarities between the replication region of the Yersinia virulence plasmid and the RepFIIA replicons.

We sequenced the minimum replication region of the virulence plasmid pYVe439-80 from a serogroup O:9 Yersinia enterocolitica. This sequence is 68% homologous on a 1,873-nucleotide stretch to the sequence of the RepFIIA replicon of the resistance plasmid R100. The sequence contains two open reading frames, repA and repB, encoding proteins of 33,478 and 9,568 daltons, respectively. The amino acid sequences of the two proteins are 77 and 55% identical, respectively, to proteins RepA1 and RepA2 of the R100 replicon. Analysis of minicells transformed with a copy number mutant demonstrated that the replication region of pYVe439-80 directs the synthesis of a 33-kilodalton protein. Disruption of repA, encoding this protein, abolished replication. Two regions of pYVe439-80 are 76 and 70% homologous, respectively, to the copy number control antisense RNA and to the origin of replication region of R100. A mutation introduced in the pYVe439-80 DNA corresponding to the R100 sequence encoding the copy number control antisense RNA resulted in an increase in copy number, indicating a functional homology between the two replicons.