A 30 S precursor of 30 S ribosomes in a mutant of Escherichia coli.
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ABSTRACT:
SUBMITTER: Markey F
PROVIDER: S-EPMC1172382 | biostudies-other | 1975 Nov
REPOSITORIES: biostudies-other
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Project description:Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that leads to the accumulation of large amounts of ribonucleoprotein ("47S") particles during exponential growth. These particles are precursors to 50S ribosomes, but are distinct from precursors detected by pulse-labelling of the parent strain and also from ribosome precursors that accumulate during inhibition of growth by CoC12. Either ribosome assembly in the mutant differs from that in the wild-type strain, or 47S particles represent a hitherto unstudied stage in the synthesis of 50S ribosomes.
| S-EPMC1172712 | biostudies-other
Project description:Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that caused the accumulation of ribonucleoprotein ('47S') particles during exponential growth. These particles are precursors to 50S ribosomes that lack three ribosomal proteins. Peptidyltransferase activity and binding at the peptidyl site of the peptidyltransferase centre are greatly decreased in 47S particles. Both these activities are lower in the 50S and 70S ribosomes of strain 15-28 than in its parent. Unusual assembly of the larger ribosomal subunit in strain 15-28 may produce completed ribosomes with diminished biological activity.
| S-EPMC1164291 | biostudies-other
Project description:Escherichia coli strain 15--28 is a mutant which during exponential growth contains large amounts of a '47S' ribonucleoprotein precursor to 50S ribosomes. The '47S particles' are more sensitive to ribonuclease than are 50S ribosomes. The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis. Isolated particles have 10--15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent. Comparison with precursor particles studied by other workers in wild-type strains of E. coli suggests that the assembly of 50S ribosomes in strain 15--28 is atypical.
| S-EPMC1163988 | biostudies-other
Project description:Pseudouridine (?) is present at conserved, functionally important regions in the ribosomal RNAs (rRNAs) from all three domains of life. Little, however, is known about the functions of ? modifications in bacterial ribosomes. An Escherichia coli strain has been constructed in which all seven rRNA ? synthases have been inactivated and whose ribosomes are devoid of all ?s. Surprisingly, this strain displays only minor defects in ribosome biogenesis and function, and cell growth is only modestly affected. This is in contrast to a strong requirement for ? in eukaryotic ribosomes and suggests divergent roles for rRNA ? modifications in these two domains.IMPORTANCE Pseudouridine (?) is the most abundant posttranscriptional modification in RNAs. In the ribosome, ? modifications are typically located at conserved, critical regions, suggesting they play an important functional role. In eukarya and archaea, rRNAs are modified by a single pseudouridine synthase (PUS) enzyme, targeted to rRNA via a snoRNA-dependent mechanism, while bacteria use multiple stand-alone PUS enzymes. Disruption of ? modification of rRNA in eukarya seriously impairs ribosome function and cell growth. We have constructed an E. coli multiple deletion strain lacking all ? modifications in rRNA. In contrast to the equivalent eukaryotic mutants, the E. coli strain is only modestly affected in growth, decoding, and ribosome biogenesis, indicating a differential requirement for ? modifications in these two domains.
| S-EPMC5786706 | biostudies-literature
Project description:1. When the methionine-requiring mutant 58-161 of Escherichia coli was starved of methionine, ribonucleic acid was made in the absence of protein synthesis. 2. Most of this ribonucleic acid was similar to that found in ribosomes but was contained in particles differing from ribosomes both in sedimentation coefficient and in chromatographic behaviour on diethylaminoethylcellulose. 3. When methionine was added to a starved culture, the ribonucleic acid synthesized during starvation was almost completely undegraded as growth resumed. A transient loss of 5-10% could be largely attributed to breakdown of messenger ribonucleic acid accumulated during starvation. 4. After the addition of methionine, ribosomes were formed from the particles, and during this period preferential synthesis of ribosomal protein took place. 5. It is suggested that under these conditions the direct synthesis of ribosomes from the particles may occur.
| S-EPMC1206783 | biostudies-other
Project description:Escherichia coli 70S ribosomes tightly bind 8 equiv of Zn(II), and EXAFS spectra indicate that Zn(II) may be protein-bound. Ribosomes were incubated with EDTA and Zn(II), and after dialysis, the resulting ribosomes bound 5 and 11 equiv of Zn(II), respectively. EXAFS studies show that the additional Zn(II) in the zinc-supplemented ribosomes binds in part to the phosphate backbone of the ribosome. Lastly, in vitro translation studies demonstrate that EDTA-treated ribosomes do not synthesize an active Zn(II)-bound metalloenzyme, while the as-isolated ribosomes do. These studies demonstrate that the majority of intracellular Zn(II) resides in the ribosome.
| S-EPMC3220600 | biostudies-literature
Project description:Escherichia coli strain 15--28 is a mutant that accumulates ribonucleoprotein ('47 S') particles during exponential growth. These particles contain mature 23 S rRNA, but lack three of the proteins of the larger ribosomal subunit, to which they are a precursor. In organisms growing at 20 degrees C, assembly of 47 S particles involves three intermediates that contain precursor 23 S rRNA, one of which has the same sedimentation properties as 47 S particles. Assembly of 50 S ribosomal subunits in the parent strain is 'normal'. There are three intermediates; each contains precursor 23 S rRNA, and one cannot be distinguished from completed subunits by sedimentation. Synthesis of 30 S ribosomal subunits in parent and mutant strains is qualitatively similar, but quantitatively different. When growth is at 37 degrees C, assembly in the mutant alters. There are now two sequential precursors to 47 S particles. Both contain precursor 23 S rRNA; one has the same sedimentation coefficient as 47 S particles. In some respects, synthesis in the mutant proceeds as though 47 S particles, rather than 50 S ribosomal subunits, are the end-product of assembly.
| S-EPMC1162074 | biostudies-other
Project description:Transcription and translation are coupled processes in bacteria. A role of transcription elongation cofactor NusG in coupling has been suggested by in vitro structural studies. NMR revealed association of the NusG carboxy-terminal domain with S10 (NusE), implying a direct role for NusG as a bridge linking RNAP and the lead ribosome. Here we present the first in vitro and in vivo evidence of full-length NusG association with mature 70S ribosomes. Binding did not require accessory factors in vitro. Mutating the NusG:S10 binding interface at NusG F165 or NusE M88 and D97 residues weakened NusG:S10 association in vivo and completely abolished it in vitro, supporting the specificity of this interaction. Mutations in the binding interface increased sensitivity to chloramphenicol. This phenotype was suppressed by rpoB*35, an RNAP mutation that reduces replisome-RNAP clashes. We propose that weakened NusG:S10 interaction leads to uncoupling when translation is inhibited, with resulting RNAP backtracking, replication blocks and formation of lethal DNA double-strand breaks.
| S-EPMC5980749 | biostudies-literature
Project description:Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in 24 Escherichia coli ribosomal proteins by tandem mass spectrometry. Detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining, and antibodies for phospho-Ser, Thr, and Tyr; or by mass spectrometry equipped with a capability to detect addition and loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, and L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given phosphorylation sites in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins.
| S-EPMC2760691 | biostudies-literature
Project description:During exponential growth, the mutatn strain Escherichia coli 15-28 accumulates 47S particles, which are unusual precursors to 50S ribosomal subunits. The 47S particles have little ability to bind chloramphenicol, but binding of a fragment of aminoacyl-tRNA is about half that by completed subunits. The 70S (and 50S) ribosomes of strain 15-28 and its parent (strain 15TP) do not differ in chloramphenicol binding. Although ribosomes from the mutant are less able than those from the parent to bind the fragment, this difference is not as marked as was found previously [Sims & Wild (1976) Biochem. J. 160, 721-726] for the binding of an analogue of peptidyl-tRNA and for peptidyltransferase activity. The altered activities may arise because strain 15-28 misassembles 50S subunits of altered conformation and because the few proteins that 47S patricles lack have vital functions in some of the partial reactions of protein synthesis.
| S-EPMC1185724 | biostudies-other