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Mathias Springer

Publications and source records attributed to Mathias Springer.

15 recordsLinked to original sources
PubMed2006

Coexistence of two protein folding states in the crystal structure of ribosomal protein L20.

The recent finding of intrinsically unstructured proteins defies the classical structure-function paradigm. However, owing to their flexibility, intrinsically unstructured proteins generally escape detailed structural investigations. Consequently little is known about the extent of conformational disorder and its role in biological functions. Here, we present the X-ray structure of the unbound ribosomal protein L20, the long basic amino-terminal extension of which has been previously interpreted as fully disordered in the absence of RNA. This study provides the first detailed picture of two protein folding states trapped together in a crystal and indicates that unfolding occurs in discrete regions of the whole protein, corresponding mainly to RNA-binding residues. The electrostatic destabilization of the long alpha-helix and a structural communication between the two L20 domains are reminiscent of those observed in calmodulin. The detailed comparison of the two conformations observed in the crystal provides new insights into the role of unfolded extensions in ribosomal assembly.

Bacterial Proteins↗
PubMed2005

Impact of ionizing radiation on the life cycle of Saccharomyces cerevisiae Ty1 retrotransposon.

Ty1 elements, LTR-retrotransposons of Saccharomyces cerevisiae, are known to be activated by genetic and environmental stress. Several DNA-damaging agents have been shown to increase both Ty1 transcription and retrotransposition. To explore further the relationship between Ty1 mobility and DNA damage, we have studied the impact of ionizing radiation at different steps of the Ty1 life cycle. We have shown that Ty1 transposition is strongly activated by gamma-irradiation and we have analysed its effect on Ty1 transcription, TyA1 protein and Ty1 cDNA levels. The activation of transposition rises with increasing doses of gamma-rays and is stronger for Ty1 elements than for the related Ty2 elements. Ty1 RNA levels are markedly elevated upon irradiation; however, no significant increase of TyA1 protein was detected as measured by TYA1-lacZ fusions and by Western blot. A moderate increase in Ty1 cDNA levels was also observed, indicating that ionizing radiation can induce the synthesis of Ty1 cDNA. In diploid cells and ste12 mutants, where both Ty1 transcription and transposition are repressed, gamma-irradiation is able to activate Ty1 transposition and increases Ty1 RNA levels. These results suggest the existence of a specific regulatory pathway involved in Ty1 response to the gamma-irradiation that would be independent of Ste12 and mating-type factors. Our findings also indicate that ionizing radiation acts on several steps of the Ty1 life cycle.

Blotting, Northern↗
PubMed2005

Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding.

The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.

Bacterial Proteins↗
PubMed2005

Double molecular mimicry in Escherichia coli: binding of ribosomal protein L20 to its two sites in mRNA is similar to its binding to 23S rRNA.

Escherichia coli ribosomal L20 is one of five proteins essential for the first reconstitution step of the 50S ribosomal subunit in vitro. It is purely an assembly protein, because it can be withdrawn from the mature subunit without effect on ribosome activity. In addition, L20 represses the translation of its own gene by binding to two sites in its mRNA. The first site is a pseudoknot formed by a base-pairing interaction between nucleotide sequences separated by more than 280 nucleotides, whereas the second site is an irregular helix formed by base-pairing between neighbouring nucleotide sequences. Despite these differences, the mRNA folds in such a way that both L20 binding sites share secondary structure similarity with the L20 binding site located at the junction between helices H40 and H41 in 23S rRNA. Using a set of genetic, biochemical, biophysical, and structural experiments, we show here that all three sites are recognized similarly by L20.

Base Sequence↗
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

Severe adenine starvation activates Ty1 transcription and retrotransposition in Saccharomyces cerevisiae.

Ty1 retrotransposons of the yeast Saccharomyces cerevisiae are activated by different kinds of stress. Here we show that Ty1 transcription is stimulated under severe adenine starvation conditions. The Bas1 transcriptional activator, responsible for the induction of genes of the de novo AMP biosynthesis pathway (ADE) in the absence of adenine, is not involved in this response. Activation occurs mainly on Ty1 elements, whose expression is normally repressed by chromatin and is suppressed in a hta1-htb1Delta mutant that alters chromatin structure. Activation is also abolished in a snf2Delta mutant. Several regions of the Ty1 promoter are necessary to achieve full activation, suggesting that full integrity of the promoter sequences might be important for activation. Together, these observations are consistent with a model in which the activation mechanism involves chromatin remodeling at Ty1 promoters. The consequence of Ty1 transcriptional activation in response to adenine starvation is an increase in Ty1 cDNA levels and a relief of Ty1 dormancy. The retrotransposition of four native Ty1 elements increases in proportion to their increase in transcription. Implications for the regulation of Ty1 mobility by changes in Ty1 mRNA levels are discussed.

Adenine↗
PubMed2005

The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control.

The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control.

Bacterial Proteins↗
PubMed2003

Bacterial translational control at atomic resolution.

Translational regulation allows rapid adaptation of protein synthesis to environmental conditions. In prokaryotes, the synthesis of many RNA-binding proteins is regulated by a translational feedback mechanism involving a competition between their natural substrate and their binding site on mRNA, which are often thought to resemble each other. This article describes the case of threonyl-tRNA synthetase, which represses the translation of its own mRNA. Recent data provide the first opportunity to describe at the atomic level both the extent and the limit of mimicry between the way this enzyme recognizes tRNA(Thr) and its regulatory site in mRNA. The data also give some clues about how the binding of the synthetase to its mRNA inhibits translation.

Base Sequence↗
PubMed2003

The modular structure of Escherichia coli threonyl-tRNA synthetase as both an enzyme and a regulator of gene expression.

In addition to its role in tRNA aminoacylation, Escherichia coli threonyl-tRNA synthetase is a regulatory protein which binds a site, called the operator, located in the leader of its own mRNA and inhibits translational initiation by competing with ribosome binding. This work shows that the two essential steps of regulation, operator recognition and inhibition of ribosome binding, are performed by different domains of the protein. The catalytic and the C-terminal domain of the protein are involved in binding the two anticodon arm-like structures in the operator whereas the N-terminal domain of the enzyme is responsible for the competition with the ribosome. This is the first demonstration of a modular structure for a translational repressor and is reminiscent of that of transcriptional regulators. The mimicry between the operator and tRNA, suspected on the basis of previous experiments, is further supported by the fact that identical regions of the synthetase recognize both the operator and the tRNA anticodon arm. Based on these results, and recent structural data, we have constructed a computer-derived molecular model for the operator-threonyl-tRNA synthetase complex, which sheds light on several essential aspects of the regulatory mechanism.

Binding Sites↗
PubMed2002

NMR structure of bacterial ribosomal protein l20: implications for ribosome assembly and translational control.

L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved. It is composed of an unstructured N-terminal domain comprising residues 1-58 and a C-terminal alpha-helical domain. This is in contrast with what is observed in the bacterial 50S subunit, where the N-terminal region folds as an elongated alpha-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The pre-folded C-terminal domain would make a primary interaction with a specific site on the 23S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.

Amino Acid Sequence↗
PubMed2002

An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes.

In Listeria monocytogenes, virulence genes are maximally expressed at 37 degrees C, almost silent at 30 degrees C and controlled by PrfA, a transcriptional activator whose expression is thermoregulated. Here, we show that the untranslated mRNA (UTR) preceding prfA, forms a secondary structure, which masks the ribosome binding region. Mutations predicted to destabilize this structure led to virulence gene expression and invasion of mammalian cells at 30 degrees C. Chemical probing, native gel electrophoresis, in vitro translation, and "compensatory" and "increased stability" mutations demonstrated that the UTR switches between a structure active at high temperatures, and another inactive at low temperatures. Strikingly, when the DNA corresponding to the UTR was fused to gfp in E. coli, bacteria became fluorescent at 37 degrees C, but not at 30 degrees C. This mechanism of posttranscriptional thermoregulation may have important applications.

Bacterial Proteins↗
PubMed2002

Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: a possible case of ribosomal RNA-messenger RNA molecular mimicry.

In addition to being a component of the large ribosomal subunit, ribosomal protein L20 of Escherichia coli also acts as a translational repressor. L20 is synthesized from the IF3 operon that contains three cistrons coding for IF3, and ribosomal proteins L35 and L20. L20 directly represses the expression of the gene encoding L35 and the expression of its own gene by translational coupling. All of the cis-acting sequences required for repression by L20, called the operator, are found on an mRNA segment extending from the middle of the IF3 gene to the start of the L35 gene. L20-mediated repression requires a long-range base-pairing interaction between nucleotide residues within the IF3 gene and residues just upstream of the L35 gene. This interaction results in the formation of a pseudoknot. Here we show that L20 causes protection of nucleotide residues in two regions of the operator in vitro. The first region is the pseudoknot itself and the second lies in an irregular stem located upstream of the L35 gene. By primer extension analysis, we show that L20 specifically induces reverse transcriptase stops in both regions. Therefore, these two regions define two L20-binding sites in the operator. Using mutations and deletions of rpml'-'lacZ fusions, we show that both sites are essential for repression in vivo. However L20 can bind to each site independently in vitro. One site is similar to the L20-binding site on 23S rRNA. Here we propose that L20 recognizes its mRNA and its rRNA in similar way.

5' Untranslated Regions↗
PubMed2002

Structural basis of translational control by Escherichia coli threonyl tRNA synthetase.

Escherichia coli threonyl-tRNA synthetase (ThrRS) represses the translation of its own messenger RNA by binding to an operator located upstream of the initiation codon. The crystal structure of the complex between the core of ThrRS and the essential domain of the operator shows that the mRNA uses the recognition mode of the tRNA anticodon loop to initiate binding. The final positioning of the operator, upon which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface. The finding of other thrS operators that have this conserved motif leads to a generalization of this regulatory mechanism to a subset of Gram-negative bacteria.

Anticodon↗
PubMed2002

Differential effects of chromatin and Gcn4 on the 50-fold range of expression among individual yeast Ty1 retrotransposons.

Approximately 30 copies of the Ty1 retrotransposon are present in the genome of Saccharomyces cerevisiae. Previous studies gave insights into the global regulation of Ty1 transcription but provided no information on the behavior of individual genomic elements. This work shows that the expression of 31 individual Ty1 elements in S288C varies over a 50-fold range. Their transcription is repressed by chromatin structures, which are antagonized by the Swi/Snf and SAGA chromatin-modifying complexes in highly expressed Ty1 elements. These elements carry five potential Gcn4 binding sites in their promoter regions that are mostly absent in weakly expressed Ty1 copies. Consistent with this observation, Gcn4 activates the transcription of highly expressed Ty1 elements only. One of the potential Gcn4 binding sites acts as an upstream activating sequence in vivo and interacts with Gcn4 in vitro. Since Gcn4 has been shown to interact with Swi/Snf and SAGA, we predict that Gcn4 activates Ty1 transcription by targeting these complexes to specific Ty1 promoters.

Amino Acids↗