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1.
mSystems ; 8(1): e0109822, 2023 02 23.
Article in English | MEDLINE | ID: mdl-36651729

ABSTRACT

Many mutations in genes for ribosomal proteins (r-proteins) and assembly factors cause cell stress and altered cell fate, resulting in congenital diseases collectively called ribosomopathies. Even though all such mutations depress the cell's protein synthesis capacity, they generate many different phenotypes, suggesting that the diseases are not due simply to insufficient protein synthesis capacity. To learn more, we investigated how the global transcriptome in Saccharomyces cerevisiae responds to reduced protein synthesis generated in two different ways: abolishing the assembly of new ribosomes and inhibiting ribosomal function. Our results showed that the mechanism by which protein synthesis is obstructed affects the ribosomal protein transcriptome differentially: ribosomal protein mRNA abundance increases during the abolition of ribosome formation but decreases during the inhibition of ribosome function. Interestingly, the ratio between mRNAs from some, but not all, pairs of paralogous ribosomal protein genes encoding slightly different versions of a given r-protein changed differently during the two types of stress, suggesting that expression of specific ribosomal protein paralogous mRNAs may contribute to the stress response. Unexpectedly, the abundance of transcripts for ribosome assembly factors and translation factors remained relatively unaffected by the stresses. On the other hand, the state of the translation apparatus did affect cell physiology: mRNA levels for some other proteins not directly related to the translation apparatus also changed differentially, though not coordinately with the r-protein genes, in response to the stresses. IMPORTANCE Mutations in genes for ribosomal proteins or assembly factors cause a variety of diseases called ribosomopathies. These diseases are typically ascribed to a reduction in the cell's capacity for protein synthesis. Paradoxically, ribosomal mutations result in a wide variety of disease phenotypes, even though they all reduce protein synthesis. Here, we show that the transcriptome changes differently depending on how the protein synthesis capacity is reduced. Most strikingly, inhibiting ribosome formation and ribosome function had opposite effects on the abundance of mRNA for ribosomal proteins, while genes for ribosome translation and assembly factors showed no systematic responses. Thus, the process by which the protein synthesis capacity is reduced contributes decisively to global mRNA composition. This emphasis on process is a new concept in understanding ribosomopathies and other stress responses.


Subject(s)
Ribosomal Proteins , Saccharomyces cerevisiae Proteins , Ribosomal Proteins/genetics , Saccharomyces cerevisiae/genetics , RNA, Messenger/genetics , Ribosomes/genetics , Saccharomyces cerevisiae Proteins/genetics
2.
Int J Mol Sci ; 23(15)2022 Jul 27.
Article in English | MEDLINE | ID: mdl-35897840

ABSTRACT

According to the classic ribosome model, developed in the 1960s and 1970s, its only function is to translate the four-letter nucleic acid code into the 20 amino acid peptide-code, while polymerizing amino acids into peptides with the help of a large complement of tRNAs and translation factors that cycle on and off the ribosome [...].


Subject(s)
Protein Biosynthesis , Ribosomes , Amino Acids/metabolism , Peptides/metabolism , RNA, Transfer/chemistry , RNA, Transfer/genetics , Ribosomes/metabolism
3.
Int J Mol Sci ; 22(13)2021 Jun 22.
Article in English | MEDLINE | ID: mdl-34206573

ABSTRACT

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the "spacer" sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3' end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5' end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5' end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5' end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.


Subject(s)
RNA, Ribosomal, 5.8S/genetics , RNA, Ribosomal, 5.8S/metabolism , DNA, Ribosomal Spacer , Endoribonucleases , Gene Expression Regulation, Fungal , Nucleic Acid Conformation , RNA Processing, Post-Transcriptional , RNA, Fungal , RNA, Ribosomal, 5.8S/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Deletion
4.
PLoS One ; 15(1): e0222479, 2020.
Article in English | MEDLINE | ID: mdl-31986150

ABSTRACT

Inhibition of the synthesis of an essential ribosomal protein (r-protein) abrogates the assembly of its cognate subunit, while assembly of the other subunit continues. Ribosomal components that are not stably incorporated into ribosomal particles due to the disrupted assembly are rapidly degraded. The 60S protein uL18/L5 is an exception and this protein accumulates extra-ribosomally during inhibition of 60S assembly. Since the r-proteins in each ribosomal subunit are essential only for the formation of their cognate subunit, it would be predicted that accumulation of extra-ribosomal uL18/L5 is specific to restriction of 60S assembly and does not occur abolition of 40S assembly. Contrary to this prediction, we report here that repression of 40S r-protein genes does lead to accumulation of uL18/L5 outside of the ribosome. Furthermore, the effect varies depending on which 40S ribosomal protein is repressed. Our results also show extra-ribosomal uL18/L5 is formed during 60S assembly, not during degradation of mature cytoplasmic 60S subunits. Finally, we propose a model for the accumulation of extra-ribosomal uL18 in response to the abolition of 40S r-proteins.


Subject(s)
Ribosomal Proteins/genetics , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/genetics , Ribosomes/genetics , Humans , RNA, Ribosomal/genetics
6.
Int J Mol Sci ; 20(11)2019 Jun 08.
Article in English | MEDLINE | ID: mdl-31181743

ABSTRACT

In eukaryotes three of the four ribosomal RNA (rRNA) molecules are transcribed as a long precursor that is processed into mature rRNAs concurrently with the assembly of ribosomal subunits. However, the relative timing of association of ribosomal proteins with the ribosomal precursor particles and the cleavage of the precursor rRNA into the subunit-specific moieties is not known. To address this question, we searched for ribosomal precursors containing components from both subunits. Particles containing specific ribosomal proteins were targeted by inducing synthesis of epitope-tagged ribosomal proteins followed by pull-down with antibodies targeting the tagged protein. By identifying other ribosomal proteins and internal rRNA transcribed spacers (ITS1 and ITS2) in the immuno-purified ribosomal particles, we showed that eS7/S7 and uL4/L4 bind to nascent ribosomes prior to the separation of 40S and 60S specific segments, while uS4/S9, uL22, and eL13/L13 are bound after, or simultaneously with, the separation. Thus, the incorporation of ribosomal proteins from the two subunits begins as a co-assembly with a single rRNA molecule, but is finished as an assembly onto separate precursors for the two subunits.


Subject(s)
Organelle Biogenesis , Protein Multimerization , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Protein Binding , RNA, Ribosomal/metabolism , Saccharomyces cerevisiae
7.
Life Sci Alliance ; 2(2)2019 04.
Article in English | MEDLINE | ID: mdl-30837296

ABSTRACT

The 1:1 balance between the numbers of large and small ribosomal subunits can be disturbed by mutations that inhibit the assembly of only one of the subunits. Here, we have investigated if the cell can counteract an imbalance of the number of the two subunits. We show that abrogating 60S assembly blocks 40S subunit accumulation. In contrast, cessation of the 40S pathways does not prevent 60S accumulation, but does, however, lead to fragmentation of the 25S rRNA in 60S subunits and formation of a 55S ribosomal particle derived from the 60S. We also present evidence suggesting that these events occur post assembly and discuss the possibility that the turnover of subunits is due to vulnerability of free subunits not paired with the other subunit to form 80S ribosomes.


Subject(s)
Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Survival/physiology , Galactokinase/genetics , Gene Expression Regulation, Fungal , Promoter Regions, Genetic , Protein Stability , RNA, Ribosomal/metabolism , RNA, Ribosomal, 18S/metabolism , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/genetics , Saccharomyces cerevisiae Proteins/genetics , Trans-Activators/genetics
8.
PLoS One ; 12(10): e0186494, 2017.
Article in English | MEDLINE | ID: mdl-29028845

ABSTRACT

Abrogation of ribosome synthesis (ribosomal stress) leads to cell cycle arrest. However, the immediate cell response to cessation of ribosome formation and the transition from normal cell proliferation to cell cycle arrest have not been characterized. Furthermore, there are conflicting conclusions about whether cells are arrested in G2/M or G1, and whether the cause is dismantling ribosomal assembly per se, or the ensuing decreased number of translating ribosomes. To address these questions, we have compared the time kinetics of key cell cycle parameters after inhibiting ribosome formation or function in Saccharomyces cerevisiae. Within one-to-two hours of repressing genes for individual ribosomal proteins or Translation Elongation factor 3, configurations of spindles, spindle pole bodies began changing. Actin began depolarizing within 4 hours. Thus the loss of ribosome formation and function is sensed immediately. After several hours no spindles or mitotic actin rings were visible, but membrane ingression was completed in most cells and Ace2 was localized to daughter cell nuclei demonstrating that the G1 stage was reached. Thus cell division was completed without the help of a contractile actin ring. Moreover, cell wall material held mother and daughter cells together resulting in delayed cell separation, suggesting that expression or function of daughter gluconases and chitinases is inhibited. Moreover, cell development changes in very similar ways in response to inhibition of ribosome formation and function, compatible with the notion that decreased translation capacity contributes to arresting the cell cycle after abrogation of ribosome biogenesis. Potential implications for the mechanisms of diseases caused by mutations in ribosomal genes (ribosomopathies) are discussed.


Subject(s)
Cell Cycle , Protein Biosynthesis , Ribosomes/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Actins/metabolism , Cell Wall/metabolism , DNA-Binding Proteins/metabolism , Peptide Elongation Factors/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/metabolism , Transcription Factors/metabolism
9.
Structure ; 25(8): 1233-1241.e3, 2017 08 01.
Article in English | MEDLINE | ID: mdl-28689968

ABSTRACT

Erythromycin is a clinically useful antibiotic that binds to an rRNA pocket in the ribosomal exit tunnel. Commonly, resistance to erythromycin is acquired by alterations of rRNA nucleotides that interact with the drug. Mutations in the ß hairpin of ribosomal protein uL22, which is rather distal to the erythromycin binding site, also generate resistance to the antibiotic. We have determined the crystal structure of the large ribosomal subunit from Deinococcus radiodurans with a three amino acid insertion within the ß hairpin of uL22 that renders resistance to erythromycin. The structure reveals a shift of the ß hairpin of the mutated uL22 toward the interior of the exit tunnel, triggering a cascade of structural alterations of rRNA nucleotides that propagate to the erythromycin binding pocket. Our findings support recent studies showing that the interactions between uL22 and specific sequences within nascent chains trigger conformational rearrangements in the exit tunnel.


Subject(s)
Bacterial Proteins/chemistry , Ribosomal Proteins/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Deinococcus/chemistry , Erythromycin/chemistry , Erythromycin/pharmacology , Mutation , Protein Binding , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism
10.
Nucleic Acids Res ; 44(12): 5798-810, 2016 07 08.
Article in English | MEDLINE | ID: mdl-27257065

ABSTRACT

Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the ONLY: sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlA(crb) pause peptide.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Organelle Biogenesis , Protein Biosynthesis , RNA-Binding Proteins/genetics , Ribosomal Proteins/genetics , Base Sequence , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Models, Molecular , Polyribosomes , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Secondary , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , RNA, Ribosomal, 23S/genetics , RNA, Ribosomal, 23S/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Sequence Deletion , Transcription Factors/genetics , Transcription Factors/metabolism
11.
Curr Opin Struct Biol ; 24: 165-9, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24524803

ABSTRACT

A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as 'new system' names.


Subject(s)
Ribosomal Proteins/classification , Terminology as Topic , Animals , Bacteria/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/classification , Fungal Proteins/chemistry , Fungal Proteins/classification , Humans , Ribosomal Proteins/chemistry , Ribosome Subunits/chemistry , Yeasts/chemistry
12.
Mol Biol Cell ; 24(23): 3620-33, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24109599

ABSTRACT

The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.


Subject(s)
Cell Cycle , Protein Biosynthesis , Ribosomal Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Cell Nucleus/metabolism , DNA, Fungal/metabolism , Flow Cytometry , Models, Biological , Phenotype , Saccharomyces cerevisiae/growth & development
13.
RNA ; 15(7): 1407-16, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19465684

ABSTRACT

RNase MRP is a nucleolar RNA-protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.


Subject(s)
Endoribonucleases/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Fungal/metabolism , RNA, Ribosomal/metabolism , Saccharomyces cerevisiae/enzymology , Base Sequence , Blotting, Northern , Endoribonucleases/antagonists & inhibitors , Endoribonucleases/genetics , Exoribonucleases/genetics , Exoribonucleases/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Phenotype , RNA, Fungal/genetics , RNA, Ribosomal/genetics , Ribonucleoproteins/antagonists & inhibitors , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Temperature , Transcription, Genetic
14.
J Bacteriol ; 190(17): 5862-9, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18586934

ABSTRACT

Amino acids are polymerized into peptides in the peptidyl transferase center of the ribosome. The nascent peptides then pass through the exit tunnel before they reach the extraribosomal environment. A number of nascent peptides interact with the exit tunnel and stall elongation at specific sites within their peptide chain. Several mutational changes in RNA and protein components of the ribosome have previously been shown to interfere with pausing. These changes are localized in the narrowest region of the tunnel, near a constriction formed by ribosomal proteins L4 and L22. To expand our knowledge about peptide-induced pausing, we performed a comparative study of pausing induced by two peptides, SecM and a short peptide, Crb(CmlA), that requires chloramphenicol as a coinducer of pausing. We analyzed the effects of 15 mutational changes in L4 and L22, as well as the effects of methylating nucleotide A2058 of 23S rRNA, a nucleotide previously implicated in pausing and located close to the L4-L22 constriction. Our results show that methylation of A2058 and most mutational changes in L4 and L22 have differential effects on pausing in response to Crb(CmlA) and SecM. Only one change, a 6-amino-acid insertion after amino acid 72 in L4, affects pausing in both peptides. We conclude that the two peptides interact with different regions of the exit tunnel. Our results suggest that either the two peptides use different mechanisms of pausing or they interact differently but induce similar inhibitory conformational changes in functionally important regions of the ribosome.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Protein Biosynthesis , RNA, Bacterial/metabolism , Ribosomes/metabolism , Binding Sites/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Methylation , Models, Biological , Mutation , Nucleic Acid Conformation , RNA Processing, Post-Transcriptional , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism
15.
RNA ; 14(3): 460-4, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18218702

ABSTRACT

The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both gram-positive and gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes.


Subject(s)
Escherichia coli/genetics , RNA, Bacterial/genetics , RNA, Fungal/genetics , RNA, Ribosomal/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Base Sequence , DNA Primers/genetics , Drug Resistance, Fungal/genetics , Erythromycin/metabolism , Erythromycin/pharmacology , Escherichia coli/drug effects , Escherichia coli/metabolism , Genes, Bacterial , Genes, Fungal , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Fungal/chemistry , RNA, Fungal/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Species Specificity
16.
Mol Microbiol ; 66(4): 1039-50, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17956547

ABSTRACT

L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated 'tentacles' that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. Mutations in the tentacles of L4 and L22 confer macrolide resistance in a variety of pathogenic and non-pathogenic bacteria. In Escherichia coli, a Lys-to-Glu mutation in L4 and a three-amino-acid deletion in the L22 had been reported. To learn more about the roles of the tentacles in ribosome assembly and function, we isolated additional erythromycin-resistant E. coli mutants. Eight new mutations mapped in L4, all within the tentacle. Two new mutations were identified in L22; one mapped outside the tentacle. Insertion mutations were found in both genes. All of the mutants grew slower than the parent, and they all showed reduced in vivo rates of peptide-chain elongation and increased levels of precursor 23S rRNA. Large insertions in L4 and L22 resulted in very slow growth and accumulation of abnormal ribosomal subunits. Our results highlight the important role of L4 and L22 in ribosome function and assembly, and indicate that a variety of changes in these proteins can mediate macrolide resistance.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Erythromycin/pharmacology , Escherichia coli/drug effects , Mutation , Ribosomal Proteins/genetics , Amino Acid Sequence , Base Sequence , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Ribosomal Proteins/metabolism
17.
J Bacteriol ; 186(17): 5933-7, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15317799

ABSTRACT

We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria.


Subject(s)
Gene Expression Regulation, Bacterial , Ribosomal Proteins/biosynthesis , Vibrio cholerae/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Base Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Order , Genes, Reporter , Molecular Sequence Data , Nucleic Acid Conformation , Operon , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomal Proteins/physiology , Vibrio cholerae/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
18.
Nucleic Acids Res ; 32(12): 3703-11, 2004.
Article in English | MEDLINE | ID: mdl-15254272

ABSTRACT

RNase MRP is an endonuclease participating in ribosomal RNA processing. It consists of one RNA and at least nine protein subunits. Using oligonucleotide-directed mutagenesis, we analyzed the functional role of five of the hairpins in the secondary structure of the RNA subunit of Saccharomyces cerevisiae RNase MRP. Deletion of an entire hairpin was either lethal or resulted in very poor growth. However, peripheral portions constituting up to 70% of a hairpin could be deleted without effects on cell growth rate or processing of rRNA. To determine whether these hairpins perform redundant functions, we analyzed mutants combining four or five benign hairpin deletions. Simultaneous removal of four of these hairpin segments had no detectable effect. Removing five created a temperature- and cold-sensitive enzyme, but these deficiencies could be partially overcome by a mutation in one of the RNase MRP protein subunits, or by increasing the copy number of several of the protein subunit genes. These observations suggest that the peripheral elements of the RNA hairpins contain no structures or sequences required for substrate recognition, catalysis or binding of protein subunits. Thus, the functionally essential elements of the RNase MRP RNA appear to be concentrated in the core of the subunit.


Subject(s)
Endoribonucleases/metabolism , RNA, Fungal/chemistry , RNA, Fungal/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae/enzymology , Base Sequence , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , RNA Stability , RNA, Fungal/genetics , RNA, Ribosomal/metabolism , Ribonucleoproteins/genetics , Saccharomyces cerevisiae/genetics , Suppression, Genetic
19.
RNA ; 9(10): 1188-97, 2003 Oct.
Article in English | MEDLINE | ID: mdl-13130133

ABSTRACT

Ribosomal proteins L4 and L22 both have a globular domain that sits on the surface of the large ribosomal subunit and an extended loop that penetrates its core. The tips of both loops contribute to the lining of the peptide exit tunnel and have been implicated in a gating mechanism that might regulate the exit of nascent peptides. Also, the extensions of L4 and L22 contact multiple domains of 23S rRNA, suggesting they might facilitate rRNA folding during ribosome assembly. To learn more about the roles of these extensions, we constructed derivatives of both proteins that lack most of their extended loops. Our analysis of ribosomes carrying L4 or L22 deletion proteins did not detect any significant difference in their sedimentation property or polysome distribution. Also, the role of L4 in autogenous control was not affected. We conclude that these extensions are not required for ribosome assembly or for L4-mediated autogenous control of the S10 operon.


Subject(s)
Escherichia coli Proteins , Escherichia coli/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal, 23S/metabolism , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Amino Acid Sequence , Binding Sites , Escherichia coli/genetics , Gene Deletion , Gene Expression Regulation, Bacterial , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Operon/genetics , RNA, Bacterial/metabolism , RNA-Binding Proteins/genetics , Ribosomal Proteins/genetics
20.
J Biol Chem ; 278(30): 28237-45, 2003 Jul 25.
Article in English | MEDLINE | ID: mdl-12738792

ABSTRACT

Ribosomal protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence. This process presumably involves L4 interaction with the leader mRNA. Here, we report direct, specific, and independent binding of ribosomal protein L4 to the S10 mRNA leader in vitro. Most of the binding energy is contributed by a small hairpin structure within the leader region, but a 64-nucleotide sequence is required for the bona fide interaction. Binding to the S10 leader mRNA is competed by the 23 S rRNA L4 binding site. Although the secondary structures of the mRNA and rRNA binding sites appear different, phosphorothioate footprinting of the L4-RNA complexes reveals close structural similarity in three dimensions. Mutational analysis of the mRNA binding site is compatible with the structural model. In vitro binding of L4 induces structural changes of the S10 leader RNA, providing a first clue for how protein L4 may provoke transcription termination.


Subject(s)
Escherichia coli/metabolism , Gene Expression Regulation, Enzymologic , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , 5' Untranslated Regions/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Binding, Competitive , Collodion/pharmacology , DNA Mutational Analysis , Dose-Response Relationship, Drug , Iodine/pharmacology , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Phylogeny , Protein Binding , Protein Structure, Secondary , RNA, Messenger/metabolism , RNA, Ribosomal, 23S/metabolism , Sequence Homology, Amino Acid , Transcription, Genetic
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