Immunological studies of Escherichia coli mutants lacking one or two ribosomal proteins

Dabbs, Eric R.; Hasenbank, Renate; Kastner, Berthold; Rak, Karl-Heinz; Wartusch, B.; Stöffler, Georg

doi:10.1007/bf00392166

Cited by 56 publications

(28 citation statements)

References 24 publications

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“…AM111, an erythromycin-independent revertant of AM, lacks ribosomal proteins S17 and L29 (21,22). AM111-5, selected as a faster-growing spontaneous derivative of AM111, lacks L29 alone (23). These observations, together with ours presented here, suggest that BSC and MC at least of etiolated maize leaves contain two different populations of functional chloroplast ribosomes with respect to L29: one class with and the other without L29.…”

Section: Discussionsupporting

confidence: 58%

Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29

Zhao¹,

Xu²,

Zucchi³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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Seedlings grown in darkness, i.e., etiolated seedlings, lack chlorophyll and most other components of the photosynthetic apparatus. On illumination, the plastids become photosynthetically competent through the production of chlorophylls and proteins encoded by certain chloroplast and nuclear genes. There are two types of photosynthetic cells in leaves of the C4 plant maize: bundle sheath cells (BSC) and adjacent mesophyll cells (MC). Some proteins of the maize photosynthetic machinery are solely or preferentially localized in MC and others in BSC. A particular gene may be photoregulated up in one cell type and down in the other. Transcripts of the nuclear gene rpl29, encoding the chloroplast ribosomal protein L29, increase in abundance about 17-fold during light-induced maturation of plastids. There is about 1.5 times more L29 protein in ribosomes of greening leaves than in ribosomes of unilluminated leaves; the L29 contents of MC and BSC are about the same. However, L21 is present about equally in plastid ribosomes of unilluminated and illuminated seedlings. In contrast to both L29 and L21, the fraction of the ribosome population containing L2 is about the same in MC and BSC of etiolated leaves but, on illumination, the proportion of the ribosome population with L2 increases in BSC but not in MC. The existence of different subpopulations of plastid ribosomes-e.g., those with and without L21 and͞or L29 during development-evokes interesting, but as yet unanswered, questions about the roles of different types of ribosomes in differentiation.When germinated and grown in darkness, angiosperm seedlings are etiolated. They lack chlorophyll and many protein components of the photosynthetic apparatus. On illumination, the required components of the photosynthetic apparatus form, and the seedlings become photosynthetically competent.Maize is a C4 plant. Certain steps in photosynthetic carbon fixation are limited to the mesophyll cells (MC) of the leaf, and other photosynthetic processes, including carbon dioxide fixation by ribulose bisphosphate carboxylase, are limited to the adjacent bundle sheath cells (BSC). MC have both of the two photosynthetic energy transducing photosystems (PSI and PSII), but BSC are poor in the oxygen evolving PSII. MC and BSC are morphologically distinct in etiolated leaves. In the course of light-induced development, genes for some components of the photosynthetic apparatus are expressed in both MC and BSC, but others are expressed differently in the two types of cells.Plastid genes whose transcript levels change on illumination of dark-grown seedlings have been identified by hybridizing mRNAs prepared from unilluminated and illuminated leaf tissues against restriction fragments of plastid DNA (e.g., 1-3). Genes for the large subunit of ribulose bisphosphate carboxylase and genes for components of the energy-transducing elements of the photosynthetic apparatus are prominent among those whose expression has been shown to be promoted on illumination of dark-grown maize seedlings.To broa...

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Section: Discussionsupporting

confidence: 58%

Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29

Zhao¹,

Xu²,

Zucchi³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…The strong growth phenotype of ⌬S9 is consistent with a partial defect in ribosome assembly. Although Dabbs and coworkers (12)(13)(14) have previously reported S9 and S13 deletion strains, the method of Link et al (15) provided a means to create precise chromosomal deletions. We did not attempt to generate an S13 deletion strain.…”

Section: Resultsmentioning

confidence: 99%

“…By contrast, omission of S13 in reconstitutions carried out with purified components yielded particles whose sedimentation was indistinguishable from WT 30S subunits, but slightly defective in functional tests (8). In early genetic screens, Dabbs and coworkers reported deletions of both S9 (12,13) and S13 (14). The S9 deletion was obtained in a search for dependence on kasugamycin, an antibiotic that inhibits binding of fMet-tRNA fMet during initiation.…”

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confidence: 99%

Creating ribosomes with an all-RNA 30S subunit P site

Hoang

Fredrick

Noller

2004

Proc. Natl. Acad. Sci. U.S.A.

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Ribosome crystal structures have revealed that two small subunit proteins, S9 and S13, have C-terminal tails, which, together with several features of 16S rRNA, contact the anticodon stem-loop of P-site tRNA. To test the functional importance of these protein tails, we created genomic deletions of the C-terminal regions of S9 and S13. All of the tail deletions, including double mutants containing deletions in both S9 and S13, were viable, showing that Escherichia coli cells can synthesize all of their proteins by using ribosomes that contain 30S P sites composed only of RNA. However, these mutants have slower growth rates, indicating that the tails may play a supporting functional role in translation. In vitro analysis shows that 30S subunits purified from the S13 deletion mutants have a generally decreased affinity for tRNA, whereas deletion of the S9 tail selectively affects the binding of tRNAs whose anticodon stem sequences are most divergent from that of initiator tRNA. E xtensive evidence from more than three decades of experiments (1) supports the idea that ribosomal function is based primarily on rRNA (2, 3). This is most clearly seen in ribosome crystal structures, which show that the functional region of the ribosome, at the subunit interface, is composed mostly of rRNA, whereas the ribosomal proteins are found mainly at the periphery of the ribosome (4-7). However, there are a number of instances where ribosomal proteins contact tRNA (6). One of these occurs in the 30S P site, which plays a major role in initiation of protein synthesis, binding the initiator tRNA and positioning the start codon of mRNA. The 30S P site is also responsible for binding and positioning the anticodon stem-loop (ASL) of peptidyl-tRNA during polypeptide elongation and for maintaining the translational reading frame when the A site is vacant. Binding of the ASL depends not only on base-pairing with the P-site codon of mRNA, but also on interactions with the 30S subunit itself. In addition to contacts involving several elements of 16S rRNA, the extended C-terminal tails of two proteins, S9 and S13, penetrate the 30S P site within contact distance of the ASL (5, 6). The universally conserved C-terminal arginine of S9 appears to contact phosphate 35 at the apex of the anticodon loop of the P-site tRNA (Fig. 1). The C-terminal tail of S9 is phylogenetically invariant in length, an observation that is explained by the crystal structure, which shows that the P-site tRNA would physically block extension of the S9 tail. The tail of S13 runs parallel to the anticodon stem, crossing the tRNA backbone between positions 29 and 30 and is thus more tolerant of variations in length (Fig. 1). Although more variable in sequence, the S13 tail always contains several basic side chains, which can make electrostatic interactions with tRNA phosphates. The interactions of the S9 and S13 protein tails with P-site tRNA and their phylogenetic conservation suggest that they are directly involved in the function of the 30S P site.Early in vitro reconsti...

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“…Since E. coli mutants lacking L27 are viable, albeit with severe impairment of growth, L27 is not absolutely essential for ribosome assembly and function (7). In contrast to this, we have classified MRP7 as an essential protein on the basis of our characterization of MRP7 null mutants, which are completely devoid of mitochondrial protein synthesis.…”

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confidence: 99%

Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of the mitochondrial ribosome.

Fearon¹,

Mason²

1988

Mol. Cell. Biol.

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The gene for MRP7, a 40-kilodalton protein of the large subunit of the yeast mitochondrial ribosome, was identified in a Agtll expression library by immunological screening with a monoclonal antibody to MRP7. An intact copy of MRP7 was then isolated from a yeast genomic library by colony hybridization. Gene disruption showed that MRP7 protein was essential for ribosomal function. Sequencing of MRP7 revealed a coding region for a basic (pI 10.6), 43.2-kilodalton protein containing 371 amino acid residues. Amino acid residues 28 to 112 of the deduced MRP7 sequence aligned with the 84 residues of the Escherichia coli ribosomal protein L27, but no significant similarity was detected between the carboxy-terminal 259 amino acids of MRP7 and other protein sequences in existing computer data bases. Within the aligned region, there was 49% amino acid identity between MRP7 and L27, compared with the 57% identity observed between L27 and its homolog in Bacilus stearothermophilus. The steady-state levels of the MRP7 protein and its mRNA were monitored in response to catabolite repression and to increased dosage of the MRP7 gene. The response to catabolite repression was characterized by a ninefold change in the level of the protein and little, if any, change in the level of the mRNA. In cells carrying the MRP7 gene on a high-copy-number plasmid, the mRNA was increased 20-fold, but there was no significant increase in MRP7 protein. Furthermore, MRP7 mRNA and protein accumulated at normal levels in [rho°] cells, which are devoid of 21S rRNA, indicating that the protein is relatively stable in the absence of ribosome assembly. Together, these results suggest that MRP7 is regulated posttranscriptionally, probably at the level of protein synthesis rather than protein turnover.Ribosomes are universal features of procaryotic and eucaryotic cells, and intensive investigation for over two decades has led to major advances in our understanding of their structure, function, synthesis, and evolution (for reviews, see references 16, 28, 31, 40, 44, and 66). Much of this knowledge has been developed in studies of the Escherichia coli ribosome, which has become the standard of comparison for the analysis of ribosomes in other organisms. In contrast to bacteria, eucaryotic cells maintain multiple, distinctly different ribosomal populations for protein synthesis in the cytoplasm, chloroplasts, and mitochondria. The presence of cytoplasmic and organellar ribosomes provides an opportunity to compare the properties of genetically separate sets of ribosomal components within a single cell and to establish relationships between these components and those of the E. coli ribosome. These comparisons should help to identify features of eucaryotic ribosomes that are special adaptations to the complex genomic organization and intracellular compartmentalization of eucaryotic cells. The ribosomes of mitochondria and chloroplasts are of special interest in this regard because the genes for the constituents of organellar ribosomes are divided between two...

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Immunological studies of Escherichia coli mutants lacking one or two ribosomal proteins

Cited by 56 publications

References 24 publications

Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29

Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29

Creating ribosomes with an all-RNA 30S subunit P site

Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of the mitochondrial ribosome.

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