The plasmid ColIb-P9 antisense Inc RNA controls expression of the RepZ replication protein and its positive regulator repY with different mechanisms.

The autonomous replication region of plasmid ColIb-P9 contains repZ encoding the RepZ replication protein, and inc and repY as the negative and positive regulators of repZ translation, respectively. inc encodes the antisense Inc RNA, and repY is a short open reading frame upstream of repZ. Translation of repY enables repZ translation by inducing formation of a pseudoknot containing stem-loop I, which base pairs with the sequence preceding the repZ start codon. Inc RNA inhibits both repY translation and formation of the pseudoknot by binding to the loop I. To investigate control of repY expression by Inc RNA, we isolated a number of mutations that express repY in the presence of Inc RNA. One class of mutations delete a part of another stem-loop (II), which derepresses repY expression by initiating translation at codon 10 (GUG), located within this structure. Point mutations in stem-loop II can also derepress repY translation, and the introduction of compensatory base-changes restores control of repY translation. These results not only indicate that suppressing a cryptic start codon by secondary structure is important for maintaining the translational control of repZ but also demonstrate that the position of start site for repY translation is critical for its control by Inc RNA. Thus, Inc RNA controls repY translation by binding in the vicinity of the start codon, in contrast to the control of repZ expression at the level of loop-loop interaction.     

Structural analysis of late intermediate complex formed between plasmid ColIb-P9 Inc RNA and its target RNA. How does a single antisense RNA repress translation of two genes at different rates?

The antisense Inc RNA encoded by the IncIalpha ColIb-P9 plasmid replicon controls the translation of repZ encoding the replication initiator and its leader peptide repY at different rates with different mechanisms. The initial loop-loop base pairing between Inc RNA and the target in the repZ mRNA leader inhibits formation of a pseudoknot required for repZ translation. A subsequent base pairing at the 5' leader of Inc RNA blocks repY translation. To delineate the molecular basis for the differential control, we analyzed the intermediate complexes formed between RepZ mRNA and Inc RNA(54), a 5'-truncated Inc RNA derivative. We found that the initial base pairing at the loops transforms into a more stable intermediate complex by its propagation in both directions. The resulting extensive base pairing indicates that the inhibition of the pseudoknot formation is established at this stage. Furthermore, the region of extensive base pairing includes bases different in related plasmids showing different incompatibility. Thus, the observed extensive base pairing is important for determining the incompatibility of the low-copy-number plasmids. We discuss the evolution of replication control systems found in IncIalpha, IncB, and IncFII group plasmids.     

Organization of the replication control region of plasmid ColIb-P9.

We identified a 1,845-base-pair sequence that contains essential information for the autonomous replication and regulation of the 93-kilobase-pair IncI alpha group ColIb-P9 plasmid. Biochemical and genetic analyses revealed that this sequence specifies at least two structural genes, designated repZ and inc. The repZ gene encodes a protein with a molecular weight of 39,000, which probably functions as an initiator for the ColIb-P9 replicon. The inc gene that phenotypically governs the incompatibility encodes an RNA with a size of about 70 bases. This small RNA acts in trans to repress the expression of repZ, thereby functioning to maintain a constant copy number of the ColIb-P9 replicon in host cells.     

Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site.

Replication of a low-copy-number IncIalpha plasmid ColIb-P9 depends on expression of the repZ gene encoding the replication initiator protein. repZ expression is negatively controlled by the small antisense Inc RNA, and requires formation of a pseudoknot in the RepZ mRNA consisting of stem-loop I, the Inc RNA target, and a downstream sequence complementary to the loop I. The loop I sequence comprises 5'-rUUGGCG-3', conserved in many prokaryotic antisense systems, and was proposed to be the important site of copy number control. Here we show that the level of repZ expression is rate-limiting for replication and thus copy number, by comparing the levels of repZ expression and copy number from different mutant ColIb-P9 derivatives defective in Inc RNA and pseudoknot formation. Kinetic analyses using in vitro transcribed RNAs indicate that Inc RNA binding and the pseudoknot formation are competitive at the level of initial base paring to loop I. This initial interaction is stimulated by the presence of the loop U residue in the 5'-rUUGGCG-3' motif. These results indicate that the competition between the two RNA-RNA interactions at the specific site is a novel regulatory mechanism for establishing the constant level of repZ expression and thus copy number.     

Structural and functional features of cis-acting sequences in the basic replicon of plasmid ColIb-P9.

We have structurally and functionally analyzed the cis-elements essential for ColIb-P9 plasmid DNA replication. The putative oriV region encompassed a region of 172 base pairs (bp) located 152 bp downstream of the repZ gene. A typical dnaA box found in this region proved nonessential for the DNA replication of ColIb-P9. The ssi signal of ColIb-P9 is a homologue of the G-sites of R1 and R100 plasmids. Deletion of the G-site led to 1.5-fold reduction of the copy number, suggesting that although this G-site is not essential, it is important for efficient ColIb-P9 DNA replication. In addition, the ColIb-P9 replicon is highly and extensively homologous with the P307 (RepFIC) replicon, and highly homologous with the R100 (RepFIIA) replicon around the G-site region. These facts imply a common ancestry from which the plasmids have evolved.     

Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.     

DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect.

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.     

A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1

Nucleotide sequences were determined for a region essential for autonomous replication and partitioning of pSC101, a plasmid whose replication is dependent on the Escherichia coli dnaA gene product. The essential replication region contains one long coding sequence, rep101 , for a protein composed of 316 amino acids, and a polypeptide approximately 37 X 10(3) Mr in size was identified as the rep101 gene product. rep101 is preceded by two inverted repeat sequences, three directly repeated sequences and a region of high A + T content containing a sequence similar to the E. coli oriC consensus sequence. Because the lesions in seven replication-deficient insertion mutants, four mutants with increased copy number and one temperature-sensitive replication mutant occur within rep101 , the rep101 gene product must control pSC101 replication and copy number. par, a region adjacent to the replication region, which functions in stable plasmid inheritance, contains several inverted repeat sequences.     

Action of plasmid ColIb-P9 on the survival after ultraviolet irradiation and on the mutagenesis of the imiC, uvm, recL, uvrE and tif1 sfiA lexA spr mutants of Escherichia coli K-12 cells

To clarify the mechanisms whereby the ColIb-P9 plasmid affects DNA repair processes, its effect was studied in mutant Escherichia coli K-12 cells with altered mutagenesis and DNA repair. The plasmid was shown to protect umuC, uvm, recL and uvrE mutants after UV irradiation. The frequency of UV-induced his+ revertants increased in the presence of the plasmid in umuC, uvm and recL mutant cells. The ColIb-P9 plasmid completely restored the UV mutability and survival of umuC mutants. These results suggest that the ColIb-P9 plasmid may encode a product similar to that of the umuC gene. In the tif1 sfiA lexA spr mutant cells where SOS functions are constitutively expressed, the ColIb-P9 plasmid increased the number of his+ revertants several times. This suggests that the action of ColIb-P9 is probably brought about not via the derepression of the recA gene but at the subsequent stages of the recA+lexA+-dependent DNA error-prone repair.     

Mutation that affects pepN translation initiation in Escherichia coli

Mutants altered in their expression of the hybrid pepN-lacZ gene have been selected for resistance to p-nitrophenyl-beta-D-thiogalactopyranoside (a bacteriostatic compound that enters the cells via lac permease). A unique mutation decreasing the level of pepN expression to 9% of that of the wild type has been studied in detail. This mutation controls in cis the expression of the pepN gene. The pepN region from a pepN-lacZ gene fusion has been cloned and sequenced. Comparison of the mutant and wild type sequences indicates that the mutation lies between the Shine-Dalgarno sequence (AGGT) and the initiation codon (AUG). This mutation is a T----C transition which might allow the formation of a stable secondary structure in the region of translation initiation thus decreasing the level of pepN expression.     

单纯疱疹病毒Ⅰ型新开放读码框UL57的鉴定

单纯疱疹病毒1型(Herpes simplex virus type 1,HSV-1)潜伏感染期间LATs的活跃转录可能与其启动子与增强子两侧的CTCF结合序列有关。本研究对位于UL56下游与LAT启动子上游之间并与CTCF结合序列重叠存在的一个新开放读码框(本研究中命名为UL57)进行了鉴定。首先利用HSV-1(F)细菌人工染色体(HSV-BAC)系统构建重组病毒HSV-EGFP-UL57,将EGFP序列插入UL57 5’端;然后分别通过Northern Blot和Western Blot检测EGFP标记的UL57的转录和表达;同时构建敲除UL57的重组病毒HSV-ΔUL57,观察UL57对病毒增殖的影响。结果显示,重组病毒HSV-EGFP-UL57感染HEp-2细胞17h后,EGFP探针检测到两条转录产物,其中1.8kb转录产物与预测大小相符;使用放线菌酮(Cycloheximide,CHX)阻断病毒即刻早期蛋白/早期蛋白合成后,UL57转录受到明显抑制。重组病毒HSV-EGFP-UL57感染Vero细胞后,9h可见融合蛋白表达,24h表达明显;融合蛋白分子量与预测大小(58kD)一致。病毒生长曲线显示,重组病毒HSV-EGFP-UL57及HSV-ΔUL57在Vero细胞中的增殖水平与HSV-1(F)基本一致。本研究表明,在HSV-1基因组(GenBank:GU734771.1)UL56下游与LAT启动子上游之间存在一个新开放读码框UL57(116 921bp～117 799bp),UL57可以进行转录,且其转录受病毒即刻早期蛋白/早期蛋白调控;转录产物可以翻译出融合蛋白,但表达水平较低。删除UL57对病毒增殖无明显影响。     

Effects of the open reading frames in the Rous sarcoma virus leader RNA on translation.

Three short open reading frames (ORFs) reside in the 5' leader of Rous sarcoma virus (RSV) and are conserved in all avian sarcoma-leukosis retroviruses. Both extensions of the lengths of the ORFs and alterations in their initiation codons affect viral replication and gene expression. To determine whether the effects on viral replication were due to translational regulation mediated by the ORFs, we examined translation following mutation of the initiation and termination codons of each of the three ORFs. We found that the ORFs marginally enhanced downstream gene expression. Moreover, repression of downstream gene translation was proportional to the lengths of the elongated ORFs and depended on the initiation contexts of the AUG codons. Although the ORFs play a major role in viral activities, their effects on translation were relatively minor. Rather, the ORFs may affect the fate of unspliced avian retroviral RNA in chronically infected cells by participating in the sorting of viral RNA for either translation or encapsidation into virions.