Biomedical subjects

M J Ruiz-Echevarría

Publications and source records attributed to M J Ruiz-Echevarría.

11 recordsLinked to original sources

PubMed2000

The RNA binding protein Pub1 modulates the stability of transcripts containing upstream open reading frames.

The nonsense-mediated mRNA decay (NMD) pathway functions to degrade transcripts containing nonsense codons. Transcripts containing mutations that insert an upstream open reading frame (uORF) in the 5'-UTR are degraded through NMD. However, several naturally occurring uORF-containing transcripts are resistant to NMD. Here we demonstrate that the GCN4 and YAP1 mRNAs, which contain uORFs, harbor a stabilizer element (STE) that prevents rapid NMD by interacting with the RNA binding protein Pub1. Conversely, a uORF-containing mRNA that lacks an STE, such as CPA1, is degraded by the NMD pathway. These results indicate that uORFs can play a pivotal role regulating both translation and turnover and that the Pub1p is a critical factor that modulates the stability of uORF-containing transcripts.

5' Untranslated Regions↗

PubMed2000

The yeast hnRNP-like protein Hrp1/Nab4 marks a transcript for nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway monitors premature translation termination and degrades aberrant mRNAs. In yeast, it has been proposed that a surveillance complex searches 3' of a nonsense codon for a downstream sequence element (DSE) associated with RNA-binding proteins. An interaction between the complex and the DSE-binding protein(s) triggers NMD. Here we describe the identification and characterization of the Hrp1/Nab4 protein as a DSE-binding factor that activates NMD. Mutations in HRP1 stabilize nonsense-containing transcripts without affecting the decay of wild-type mRNAs. Hrp1p binds specifically to a DSE-containing RNA and interacts with Upf1p, a component of the surveillance complex. A mutation in HRP1 that stabilizes nonsense-containing mRNAs abolishes its affinity for the DSE and fails to interact with Upf1p. We present a model describing how Hrp1p marks a transcript for rapid decay.

Biological Transport↗

PubMed1998

The upf3 protein is a component of the surveillance complex that monitors both translation and mRNA turnover and affects viral propagation.

The nonsense-mediated mRNA decay pathway functions to degrade aberrant mRNAs that contain premature translation termination codons. In Saccharomyces cerevisiae, the Upf1, Upf2, and Upf3 proteins have been identified as trans-acting factors involved in this pathway. Recent results have demonstrated that the Upf proteins may also be involved in maintaining the fidelity of several aspects of the translation process. Certain mutations in the UPF1 gene have been shown to affect the efficiency of translation termination at nonsense codons and/or the process of programmed -1 ribosomal frameshifting used by viruses to control their gene expression. Alteration of programmed frameshift efficiencies can affect virus assembly leading to reduced viral titers or elimination of the virus. Here we present evidence that the Upf3 protein also functions to regulate programmed -1 frameshift efficiency. A upf3-Delta strain demonstrates increased sensitivity to the antibiotic paromomycin and increased programmed -1 ribosomal frameshift efficiency resulting in loss of the M1 virus. Based on these observations, we hypothesize that the Upf proteins are part of a surveillance complex that functions to monitor translational fidelity and mRNA turnover.

Alleles↗

PubMed1998

Identifying the right stop: determining how the surveillance complex recognizes and degrades an aberrant mRNA.

The nonsense-mediated mRNA decay (NMD) pathway functions by checking whether translation termination has occurred prematurely and subsequently degrading the aberrant mRNAs. In Saccharomyces cerevisiae, it has been proposed that a surveillance complex scans 3' of the premature termination codon and searches for the downstream element (DSE), whose recognition by the complex identifies the transcript as aberrant and promotes its rapid decay. The results presented here suggest that translation termination is important for assembly of the surveillance complex. Neither the activity of the initiation ternary complex after premature translation termination has occurred nor the elongation phase of translation are essential for the activity of the NMD pathway. Once assembled, the surveillance complex is active for searching and recognizing a DSE for approximately 200 nt 3' of the stop codon. We have also identified a stabilizer sequence (STE) in the GCN4 leader region that inactivates the NMD pathway. Inactivation of the NMD pathway, as a consequence of either the DSE being too far from a stop codon or the presence of the STE, can be circumvented by inserting sequences containing a new translation initiation/termination cycle immediately 5' of the DSE. Further, the results indicate that the STE functions in the context of the GCN4 transcript to inactivate the NMD pathway.

Codon↗

PubMed1998

Replication and control of circular bacterial plasmids.

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3'-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can "sense" and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.

Base Sequence↗

PubMed1995

Translational coupling and limited degradation of a polycistronic messenger modulate differential gene expression in the parD stability system of plasmid R1.

The parD stability system of plasmid R1 is an auto-regulated operon containing two genes, kis and kid, that code, respectively, for a killer protein (Kid) and for an antagonist of Kid action (Kis protein). A polycistronic transcript and a shorter mRNA, coding only for Kis and ending in a stem-loop sequence, have been identified as the main parD transcripts in cells carrying a derepressed parD operon. In this communication we show that both parD mRNAs have a half-life close to 1 min and are present in similar amounts. Using an assay based on cell-free extracts of Escherichia coli, we demonstrate that the short kis mRNA originates from limited degradation of the bicistronic parD transcript and that the stem-loop structure within the 5' end of the kid gene is specifically required for the formation of this short transcript. In vivo experiments show that synthesis of Kis is required for efficient synthesis of Kid. These data indicate that RNA processing and translational coupling are important mechanisms that modulate the differential expression of the two genes, kis and kid, in the bicistronic parD operon.

Bacterial Proteins↗

PubMed1995

A mutation that decreases the efficiency of plasmid R1 replication leads to the activation of parD, a killer stability system of the plasmid.

The silent parD (kis/kid) stability operon of plasmid R1 is normally repressed by the co-ordinated action of the Kis and Kid proteins. In this report it is shown that a mutation in repA, the gene of the plasmid replication protein, that reduces two-fold the copy number of the plasmid, leads to the derepression of the parD system. This derepression can be prevented by a suppressor mutation in copB, a copy number control gene of plasmid R1, that increases the efficiency of replication of the repA mutant. Derepression of the wild-type parD system leads to high plasmid stability. These data show the activation of a plasmid stability operon by a mutation that reduces the efficiency of wild-type plasmid replication.

Bacterial Proteins↗

PubMed1995

Kid, a small protein of the parD stability system of plasmid R1, is an inhibitor of DNA replication acting at the initiation of DNA synthesis.

The Kid and Kis proteins are the killer component and the antagonist belonging to parD, a killer stability system of plasmid R1. The Kid and Kis proteins have been purified, the second one as a C-LYT-Kis fusion that conserves the antagonistic activity of the Kis protein, but not its auto-regulatory potential. Kid inhibits in vitro replication of CoEl to a basal level without altering the superhelicity of the template but it does not substantially affect in vitro replication of P4, a DnaA, DnaB, DnaC and DnaG-independent replicon. Kid inhibits lytic induction of a lambda, prophage, but this inhibition can be neutralized by excess DnaB. In addition, a multicopy dnaB recombinant, but not a multicopy dnaG recombinant, prevents the toxicity associated with this protein. Inhibition of ColE1 replication by Kid in vitro is prevented by the C-LYT-Kis protein. Functional analysis indicates that the antagonistic activity of Kis is independent of its activity as a co-regulator of the parD promoter. It is also shown that C-LYT-Kis and Kid interact, forming a tight complex. These results strongly suggest that the toxicity of the kid protein is due to inhibition of DnaB-dependent DNA replication, and that direct protein-protein interactions are involved in the neutralization of the activity of the killer protein by the antagonist.

Bacterial Proteins↗

PubMed1995

Transcription of repA, the gene of the initiation protein of the Pseudomonas plasmid pPS10, is autoregulated by interactions of the RepA protein at a symmetrical operator.

Transcription of the repA gene of the Pseudomonas plasmid pPS10 is initiated from a sigma 70 type promoter located 81 bp upstream from the repA gene, extends through the repA gene and the adjacent open reading frame, and ends 1114 nucleotides downstream. The repA promoter is repressed by interactions of the RepA protein with a region of 44 bp that extends from the -10 box of the promoter to the dnaA box of the origin of replication. The core of the repA operator region is formed by two in-phase invertedly repeated sequences of 8 bp, S1 and S2, that flank the -35 box of the promoter, and that share homology with the internal sequences of the iterons present in the origin of replication. RepA enters at the operator region first by protein-DNA interactions and subsequently by protein-protein interactions. These sequential interactions lead to the formation of high, medium and low-mobility electrophoretic complexes. Formation of the high-order complexes seems to be important for an efficient repression of the promoter. Interactions of RepA with the repA promoter region (repPO) occur more efficiently than with the origin of replication.

Bacterial Proteins↗

PubMed1991

Structural and functional comparison between the stability systems ParD of plasmid R1 and Ccd of plasmid F.

The stability determined by the systems ParD of plasmid R1 and Ccd of plasmid F is due to the concerted action of two proteins, a cytotoxin and an antagonist of this function. In this paper we report that CcdA and Kis proteins, the antagonists of the Ccd and ParD systems respectively, share significant sequence homologies at both ends. In Kis, these regions seem to correspond to two different domains. Despite the structural similarities, Kis and CcdA are not interchangeable. In addition we have shown that the cytotoxins of these systems, the Kid and CcdB proteins, do not share structural homologies. In contrast to CcdB, the Kid protein of the ParD system induces RecA-dependent cleavage of the cI repressor of bacteriophage lambda very inefficiently or not at all. The functional implications of these results are discussed.

Amino Acid Sequence↗

PubMed1991

The kis and kid genes of the parD maintenance system of plasmid R1 form an operon that is autoregulated at the level of transcription by the co-ordinated action of the Kis and Kid proteins.

Stability mediated by the parD system of plasmid R1 is modulated by a killer protein, Kid, and by an antagonist of this function, Kis. Determination of the 5' ends of ParD transcripts, revealed that the genes coding for these proteins are transcribed from a single promoter. Analysis of the 3' end of the ParD RNAs indicated the existence of two transcripts: one of them coding for the Kis and Kid proteins, and the other coding only for Kis. Analysis of the effects of parD+ and kis+ recombinants on the beta-galactosidase levels expressed by different transcriptional and translational parD-lacZ fusions, and on the ParD RNA levels determined by a derepressed parD mutant, indicated that the Kis and Kid proteins repress coordinately the parD system at the transcriptional level. We discuss the relevance of these results in terms of the activities of the Kis and Kid proteins and in the context of the stabilization mediated by parD.

Bacterial Proteins↗