RESUMEN
Bacterial riboswitches are molecular structures that play a crucial role in controlling gene expression to maintain cellular balance. The Escherichia coli lysC riboswitch has been previously shown to regulate gene expression through translation initiation and mRNA decay. Recent research suggests that lysC gene expression is also influenced by Rho-dependent transcription termination. Through a series of in silico, in vitro, and in vivo experiments, we provide experimental evidence that the lysC riboswitch directly and indirectly modulates Rho transcription termination. Our study demonstrates that Rho-dependent transcription termination plays a significant role in the cotranscriptional regulation of lysC expression. Together with previous studies, our work suggests that lysC expression is governed by a lysine-sensing riboswitch that regulates translation initiation, transcription termination, and mRNA degradation. Notably, both Rho and RNase E target the same region of the RNA molecule, implying that RNase E may degrade Rho-terminated transcripts, providing a means to selectively eliminate these incomplete messenger RNAs. Overall, this study sheds light on the complex regulatory mechanisms used by bacterial riboswitches, emphasizing the role of transcription termination in the control of gene expression and mRNA stability.
Asunto(s)
Riboswitch , Riboswitch/genética , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Transcripción Genética , Bacterias/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/metabolismoRESUMEN
Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.
Asunto(s)
Cobamidas , Proteínas de Escherichia coli , Escherichia coli , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano , ARN Mensajero , Riboswitch , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Cobamidas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN Helicasas/genética , ARN Helicasas/metabolismo , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas de la Membrana Bacteriana Externa , Polirribonucleótido Nucleotidiltransferasa , Proteínas de Transporte de MembranaRESUMEN
The bacterial ribosomal 5S rRNA-binding protein L5 is universally conserved (uL5). It contains the so-called P-site loop (PSL), which contacts the P-site tRNA in the ribosome. Certain PSL mutations in yeast are lethal, suggesting that the loop plays an important role in translation. In this work, for the first time, a viable Escherichia coli strain was obtained with the deletion of the major part of the PSL (residues 73-80) of the uL5 protein. The deletion conferred cold sensitivity and drastically reduced the growth rate and overall protein synthesizing capacity of the mutant. Translation rate is decreased in mutant cells as compared to the control. At the same time, the deletion causes increased levels of -1 frameshifting and readthrough of all three stop codons. In general, the results show that the PSL of the uL5 is required for maintaining both the accuracy and rate of protein synthesis in vivo.
Asunto(s)
Dominio AAA , Ribosomas , Ribosomas/genética , Codón de Terminación , Escherichia coli/genética , Saccharomyces cerevisiaeRESUMEN
Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/fisiología , Ribosomas/metabolismo , Sitios de Unión , Microscopía por Crioelectrón/métodos , Proteínas de Escherichia coli/genética , Modelos Moleculares , Unión Proteica , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/ultraestructura , Ribosomas/ultraestructuraRESUMEN
Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5' untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA-protein or mRNA-ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5' ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features.
Asunto(s)
Proteínas de Escherichia coli/fisiología , Escherichia coli/fisiología , Biosíntesis de Proteínas/fisiología , Pliegue del ARN/fisiología , ARN Mensajero/fisiología , Proteínas Ribosómicas/fisiología , Ribosomas/fisiología , Regulación Bacteriana de la Expresión Génica/fisiologíaRESUMEN
Escherichia coli NusA and NusB proteins bind specific sites, such as those in the leader and spacer sequences that flank the 16S region of the ribosomal RNA transcript, forming a complex with RNA polymerase that suppresses Rho-dependent transcription termination. Although antitermination has long been the accepted role for Nus factors in rRNA synthesis, we propose that another major role for the Nus-modified transcription complex in rrn operons is as an RNA chaperone insuring co-ordination of 16S rRNA folding and RNase III processing that results in production of proper 30S ribosome subunits. This contrarian proposal is based on our studies of nusA and nusB cold-sensitive mutations that have altered translation and at low temperature accumulate 30S subunit precursors. Both phenotypes are suppressed by deletion of RNase III. We argue that these results are consistent with the idea that the nus mutations cause altered rRNA folding that leads to abnormal 30S subunits and slow translation. According to this idea, functional Nus proteins stabilize an RNA loop between their binding sites in the 5' RNA leader and on the transcribing RNA polymerase, providing a topological constraint on the RNA that aids normal rRNA folding and processing.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factores de Elongación de Péptidos/metabolismo , ARN Ribosómico 16S/biosíntesis , Ribonucleasa III/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Eliminación de Gen , Modelos Biológicos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Conformación de Ácido Nucleico , Factores de Elongación de Péptidos/genética , Subunidades Ribosómicas Pequeñas/metabolismo , Temperatura , Factores de Transcripción/genética , Factores de Elongación TranscripcionalRESUMEN
In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained.
Asunto(s)
Proteínas de Escherichia coli/fisiología , Proteínas Ribosómicas/fisiología , Subunidades Ribosómicas Grandes Bacterianas/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Eliminación de Gen , Modelos Moleculares , ARN Ribosómico 5S/química , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Ribosomas/metabolismoRESUMEN
Ribosome biogenesis is a fundamental and multistage process. The basic steps of ribosome assembly are the transcription, processing, folding, and modification of rRNA; the translation, folding, and modification of r-proteins; and consecutive binding of ribosomal proteins to rRNAs. Ribosome maturation is facilitated by biogenesis factors that include a broad spectrum of proteins: GTPases, RNA helicases, endonucleases, modification enzymes, molecular chaperones, etc. The ribosome assembly factors assist proper rRNA folding and protein-RNA interactions and may sense the checkpoints during the assembly to ensure correct order of this process. Inactivation of these factors is accompanied by severe growth phenotypes and accumulation of immature ribosomal subunits containing unprocessed rRNA, which reduces overall translation efficiency and causes translational errors. In this review, we focus on the structural and biochemical analysis of the 30S ribosomal subunit assembly factors RbfA, YjeQ (RsgA), Era, KsgA (RsmA), RimJ, RimM, RimP, and Hfq, which take part in the decoding-center folding.
RESUMEN
We consider deterministic fast-slow dynamical systems on R m × Y of the form x k + 1 ( n ) = x k ( n ) + n - 1 a ( x k ( n ) ) + n - 1 / α b ( x k ( n ) ) v ( y k ) , y k + 1 = f ( y k ) , where α ∈ ( 1 , 2 ) . Under certain assumptions we prove convergence of the m-dimensional process X n ( t ) = x â n t â ( n ) to the solution of the stochastic differential equation d X = a ( X ) d t + b ( X ) â d L α , where L α is an α -stable Lévy process and â indicates that the stochastic integral is in the Marcus sense. In addition, we show that our assumptions are satisfied for intermittent maps f of Pomeau-Manneville type.
RESUMEN
A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild-type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.
Asunto(s)
Escherichia coli/metabolismo , Biosíntesis de Proteínas , ARN Bacteriano/metabolismo , ARN Ribosómico 5S/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Supervivencia Celular , Clonación Molecular , Escherichia coli/genética , Escherichia coli/fisiología , Proteínas de Escherichia coli/genética , Mutación , ARN Bacteriano/genética , Proteínas Represoras/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Factores de Transcripción/genéticaRESUMEN
L1 is a conserved protein of the large ribosomal subunit. This protein binds strongly to the specific region of the high molecular weight rRNA of the large ribosomal subunit, thus forming a conserved flexible structural element--the L1 stalk. L1 protein also regulates translation of the operon that comprises its own gene. Crystallographic data suggest that L1 interacts with RNA mainly by means of its domain I. We show here for the first time that the isolated domain I of the bacterial protein L1 of Thermus thermophilus and Escherichia coli is able to incorporate in vivo into the E. coli ribosome. Furthermore, domain I of T. thermophilus L1 can regulate expression of the L1 gene operon of Archaea in the coupled transcription-translation system in vitro, as well as the intact protein. We have identified the structural elements of domain I of the L1 protein that may be responsible for its regulatory properties.
Asunto(s)
Proteínas Bacterianas/química , Operón/genética , ARN Bacteriano/química , Proteínas Ribosómicas/química , Ribosomas/química , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Escherichia coli/química , Escherichia coli/genética , Datos de Secuencia Molecular , Plásmidos , Estructura Terciaria de Proteína , ARN Bacteriano/genética , ARN Ribosómico 23S/química , ARN Ribosómico 23S/genética , Proteínas Ribosómicas/genética , Resonancia por Plasmón de Superficie , Thermus thermophilus/química , Thermus thermophilus/genéticaRESUMEN
Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37 degrees C this DeltarpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the DeltarpsO strain at 37 degrees C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized.
Asunto(s)
Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel Bidimensional , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Subunidades de Proteína , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Proteínas Ribosómicas/aislamiento & purificaciónRESUMEN
Analysis of the structures of two complexes of 5 S rRNA with homologous ribosomal proteins, Escherichia coli L25 and Thermus thermophilus TL5, revealed that amino acid residues interacting with RNA can be divided into two different groups. The first group consists of non-conserved residues, which form intermolecular hydrogen bonds accessible to solvent. The second group, comprised of strongly conserved residues, form intermolecular hydrogen bonds that are shielded from solvent. Site-directed mutagenesis was used to introduce mutations into the RNA-binding site of protein TL5. We found that replacement of residues of the first group does not influence the stability of the TL5.5 S rRNA complex, whereas replacement of residues of the second group leads to destabilization or disruption of the complex. Stereochemical analysis shows that the replacements of residues of the second group always create complexes with uncompensated losses of intermolecular hydrogen bonds. We suggest that these shielded intermolecular hydrogen bonds are responsible for the recognition between the protein and RNA.