Biomedical subjects

Eliane Hajnsdorf

Publications and source records attributed to Eliane Hajnsdorf.

10 recordsLinked to original sources

PubMed2006

Hfq variant with altered RNA binding functions.

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS-lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq-RNA interaction but has probably no effect on the folding and the oligomerization of the protein.

Amino Acid Sequence↗

PubMed2005

Fate of mRNA extremities generated by intrinsic termination: detailed analysis of reactions catalyzed by ribonuclease II and poly(A) polymerase.

In all living cells 3' ends of RNA are posttranscriptionally elongated or shortened by nucleotidyl transferases and ribonucleases. The detailed analysis of the rpsO mRNA of Escherichia coli presented here demonstrates that transcription terminates in vivo at two sites located seven and eight nucleotides downstream from the GC-rich hairpin of the intrinsic terminator and that primary transcripts can be shortened by RNase II. The shortest RNA identified in the cell result from nibbling of primary transcripts. Primary transcripts and nibbled molecules can also be adenylated by poly(A) polymerase I (PAP I). In addition, kinetics of decay performed in vitro demonstrate that RNase II rapidly degrades poly(A) tails longer than 7-8 As processively while it slowly nibbles shorter tails and non adenylated RNAs distributively. Comparison of the kinetics of nibbling of oligoadenylated rpsO mRNA in vivo and in vitro lead us to conclude that the rates of shortening and elongation of the oligo(A) tails detected in vivo are very slow: about 0.5-7 nucleotides per min. We finally speculate that the slowness of oligo(A) synthesis may explain why polyadenylation does not affect the stability of mRNAs whose degradation is controlled by RNase E.

Animals↗

PubMed2005

Characterization of the molecular mechanisms involved in the differential production of erythrose-4-phosphate dehydrogenase, 3-phosphoglycerate kinase and class II fructose-1,6-bisphosphate aldolase in Escherichia coli.

A gapA-pgk gene tandem coding the glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase, is most frequently found in bacteria. However, in Enterobacteriaceae, gapA is replaced by an epd open reading frame (ORF) coding an erythrose-4-phosphate dehydrogenase and an fbaA ORF coding the class II fructose-1,6-bisphosphate aldolase follows pgk. Although epd expression is very low in Escherichia coli, we show that, in the presence of glucose, the 3 epd, pgk and fbaA ORFs are efficiently cotranscribed from promoter epd P0. Conservation of promoter epd P0 is likely due to its important role in modulation of the metabolic flux during glycolysis and gluconeogenesis. As a consequence, we found that the epd translation initiation region and ORF have been adapted in order to limit epd translation and to create an efficient RNase E entry site. We also show that fbaA is cotranscribed with pgk, from promoter epd P0 or an internal pgk P1 promoter of the extended -10 class. The differential expression of pgk and fbaA also depends upon an RNase E segmentation process, leading to individual mRNAs with different stabilities. The secondary structures of the RNA regions containing the RNase E sites were experimentally determined which brings important information on the structural features of RNase E ectopic sites.

Aldehyde Oxidoreductases↗

PubMed2005

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails.

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).

Escherichia coli Proteins↗

PubMed2004

The C-terminal domain of Escherichia coli Hfq increases the stability of the hexamer.

The Hfq (Host factor 1) polypeptide is a nucleic acid binding protein involved in the synthesis of many polypeptides. Hfq particularly affects the translation and the stability of several RNAs. In an earlier study, the use of fold recognition methods allowed us to detect a relationship between Escherichia coli Hfq and the Sm topology. This topology was further validated by a series of biophysical studies and the Hfq structure was modelled on an Sm protein. Hfq forms a beta-sheet ring-shaped hexamer. As our previous study predicted a large number of alternative conformations for the C-terminal region, we have determined whether the last 19 C-terminal residues are necessary for protein function. We find that the C-terminal truncated protein is fully capable of binding a polyadenylated RNA (K(d) of 120 pm vs. 50 pm for full-length Hfq). This result shows that the functional core of E. coli Hfq resides in residues 1-70 and confirms previous genetic studies. Using equilibrium unfolding studies, however, we find that full-length Hfq is 1.8 kcal x mol(-1) more stable than its truncated variant. Electron microscopy analysis of both truncated and full-length proteins indicates a structural rearrangement between the subunits upon truncation. This conformational change is coupled to a reduction in beta-strand content, as determined by Fourier transform infra-red. On the basis of these results, we propose that the C-terminal domain could protect the interface between the subunits and stabilize the hexameric Hfq structure. The origin of this C-terminal domain is also discussed.

Amino Acid Sequence↗

PubMed2003

The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation.

The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high specificity for a 100-nucleotide RNA harboring 18 3'-terminal A-residues. Structural analysis of the adenylated RNA-Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a single-stranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3'-5' exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.

Base Sequence↗

PubMed2003

Hfq affects the length and the frequency of short oligo(A) tails at the 3' end of Escherichia coli rpsO mRNAs.

Polyadenylation plays an important role in RNA degradation in bacterial cells. In Escherichia coli, exoribonucleases, mostly RNase II and polynucleotide phosphorylase, antagonize the synthesis of poly(A) tails by poly(A) polymerase I (PAP I). In accordance with earlier observations showing that only a small fraction of bacterial RNA is polyadenylated, we demonstrate here that approximately 10% of rpsO mRNA harbors short oligo(A) tails ranging from one to five A residues in wild-type cells. We also compared the length, frequency and distribution of poly(A) tails at the 3'-end of rpsO transcripts in vivo in the presence and absence of Hfq, a host factor that in vitro stimulates the activity of PAP I, and found that Hfq affects all three parameters. In the hfq(+) strain the average length of oligo(A) tails and frequency of polyadenylated transcripts was higher than in the hfq(-) strain and a smaller proportion of tails was found at the 3' end of transcripts terminated at the Rho- independent terminator. Our data led us to the conclusion that Hfq is involved in the recognition of 3' RNA extremities by PAP I.

3' Untranslated Regions↗

PubMed2002

Structural Modelling of the Sm-like Protein Hfq from Escherichia coli.

The Hfq polypeptide of Escherichia coli is a nucleic acid-binding protein involved in the expression of many proteins. Derivation of its three-dimensional structure is important for our understanding of its role in gene regulation at the molecular level. In this study, we combined computational and biophysical analysis to derive a possible structure for Hfq. As a first step towards determining the structure, we searched for possible sequence-structure compatibility, using secondary structure prediction and protein domain and fold-recognition methods available on the WEB. One fold, essentially beta sheet in character, the Sm motif of small nuclear ribonucleoproteins, even though it initially fell well below the confidence thresholds, was proposed and further validated by a series of biophysical and biochemical studies. The Hfq hexamer structure was modelled on the human Sm D3B structure using optimised sequence alignments and molecular mechanics methods. This structure accounts for the physico-chemical properties of Hfq and highlights amino acid residues that could interact with RNA.

Amino Acid Sequence↗

PubMed2002

RNase E and polyadenyl polymerase I are involved in maturation of CI RNA, the P4 phage immunity factor.

Bacteriophage P4 immunity is controlled by a small stable RNA (CI RNA) that derives from the processing of primary transcripts. In previous works, we observed that the endonuclease RNase P is required for the maturation of CI RNA 5'-end; moreover, we found that polynucleotide phosphorylase (PNPase), a 3' to 5' RNA-degrading enzyme, is required for efficient 5'-end processing of CI RNA, suggesting that 3'-end degradation of the primary transcript might be involved in the production of proper RNase P substrates. Here, we demonstrate that another Escherichia coli nuclease, RNase E, would appear to be involved in this process. We found that transcripts of the P4 immunity region are modified by the post-transcriptional addition of short poly(A) tails and heteropolymeric tails with prevalence of A residues. Most oligoadenylated transcripts encompass the whole cI locus and are thus compatible as intermediates in the CI RNA maturation pathway. On the contrary, in a polynucleotide phosphorylase (PNPase)-defective host, adenylation occurred most frequently within cI, implying that such transcripts are targeted for degradation. We did not find polyadenylation in a pcnB mutant, suggesting that the pcnB-encoded polyadenyl polymerase I (PAP I) is the only enzyme responsible for modification of P4 immunity transcripts. Maturation of CI RNA 5'-end in such a mutant was impaired, further supporting the idea that processing of the 3'-end of primary transcripts is an important step for efficient maturation of CI RNA by RNase P.

Base Sequence↗

PubMed1996

Escherichia coli RNase II: characterization of the promoters involved in the transcription of rnb.

The rnb gene encodes ribonuclease II (RNase II), one of the two major Escherichia coli exonucleases involved in mRNA degradation. In this paper, the rnb transcript is characterized regarding its promoter and terminator regions. The combined results from S1 nuclease protection analysis, DNase I footprinting and gene fusions with lacZ have shown that rnb is expressed from two promoters. S1 nuclease protection analysis and DNA footprinting have shown that rnb has two promoters, P1 and P2. Transcriptional and translational lacZ reporter fusions, constructed to the rnb gene, revealed that P2, the rnb proximal promoter, is stronger than P1. However, P2 is not transcribed in vitro, suggesting that an additional factor is required in vivo. The 3' end of the rnb transcript mapped to a stem-loop structure immediately after the translated region.

Amino Acid Sequence↗