Ribosomal Protein L9 Interactions with 23 S rRNA: The Use of a Translational Bypass Assay to Study the Effect of Amino Acid Substitutions

Adamski, Frances M.; Atkins, John F.; Gesteland, Raymond F.

doi:10.1006/jmbi.1996.0469

Cited by 45 publications

(57 citation statements)

References 27 publications

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“…1) (17)(18)(19). Although the positioning of L9 in crystal structures implies a rigid conformation, chemical footprinting and cross-linking experiments suggest that L9 is dynamic and may engage portions of the L1 stalk RNA and also surrounding regions of the large subunit (20,21). In support of this idea, structural biologists recently demonstrated that L9 might be inadvertently stabilized in ribosome crystals through interribosomal contacts.…”

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confidence: 99%

Crippling the Essential GTPase Der Causes Dependence on Ribosomal Protein L9

Naganathan

Moore

2013

J Bacteriol

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Ribosomal protein L9 is a component of all eubacterial ribosomes, yet deletion strains display only subtle growth defects. Although L9 has been implicated in helping ribosomes maintain translation reading frame and in regulating translation bypass, no portion of the ribosome-bound protein seems capable of contacting either the peptidyltransferase center or the decoding center, so it is a mystery how L9 can influence these important processes. To reveal the physiological roles of L9 that have maintained it in evolution, we identified mutants of Escherichia coli that depend on L9 for fitness. In this report, we describe a class of L9-dependent mutants in the ribosome biogenesis GTPase Der (EngA/YphC). Purified mutant proteins were severely compromised in their GTPase activities, despite the fact that the mutations are not present in GTP hydrolysis sites. Moreover, although L9 and YihI complemented the slow-growth der phenotypes, neither factor could rescue the GTPase activities in vitro. Complementation studies revealed that the N-terminal domain of L9 is necessary and sufficient to improve the fitness of these Der mutants, suggesting that this domain may help stabilize compromised ribosomes that accumulate when Der is defective. Finally, we employed a targeted degradation system to rapidly deplete L9 from a highly compromised der mutant strain and show that the L9-dependent phenotype coincides with a cell division defect.

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confidence: 99%

Crippling the Essential GTPase Der Causes Dependence on Ribosomal Protein L9

Naganathan

Moore

2013

J Bacteriol

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“…The exception is the mutant G24D (rplI2) in the N-terminal domain. This amino acid substitution is at the same position as the mutations G24A and G24P isolated earlier (1). Although the latter two alterations did not induce bypassing, purified L9 protein containing G24P bound poorly to a 23S rRNA fragment, which suggests that such altered L9 is not assembled into ribosomes.…”

Section: Vol 189 2007mentioning

confidence: 62%

“…The functional aspect of L9 has been studied only by analyzing how its various alterations influence the translational bypassing of an mRNA encoded from a multicopy plasmid containing the bacteriophage T4 gene 60 site (15). Although the first mutant (S93F) isolated was a genomic mutant (14), all other characterized mutants were obtained after mutagenesis of the rplI gene located on a plasmid (1). Such an approach forces the mutant form to compete with the wild-type protein encoded by the chromosomal allele rplI ϩ .…”

Section: Discussionmentioning

confidence: 99%

“…The unbiased selection for frameshift suppressors in the rplI area on the chromosome allowed isolation of rplI mutants with unchanged gene expression levels and producing a homogeneous ribosome population containing only mutated or no protein L9. Hence, the approach taken in the present study is distinctly different from that used by Adamski et al (1). Mutant analysis in the present study relied on the ability of the altered L9 to induce frameshifting on a chromosomally located ϩ1 frameshift mutation (hisC3072), a very sensitive system to detect low levels of frameshifting.…”

Section: Vol 189 2007mentioning

confidence: 99%

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Alterations in the Two Globular Domains or in the Connecting α-Helix of Bacterial Ribosomal Protein L9 Induces +1 Frameshifts

Leipuviene

Björk

2007

J Bacteriol

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The ribosomal 50S subunit protein L9, encoded by the gene rplI, is an elongated protein with an ␣-helix connecting the N-and C-terminal globular domains. We isolated rplI mutants that suppress the ؉1 frameshift mutation hisC3072 in Salmonella enterica serovar Typhimurium. These mutants have amino acid substitutions in the N-terminal domain (G24D) or in the C-terminal domain (I94S, A102D, G126V, and F132S) of L9. In addition, different one-base deletions in rplI altered either the final portion of the C terminus or removed the C-terminal domain with or without the connecting ␣-helix. An alanine-to-proline substitution at position 59 (A59P), which breaks the ␣-helix between the globular domains, induced ؉1 frameshifting, suggesting that the geometrical relationship between the N and C domains is important to maintain the reading frame. Except for the alterations G126V in the C terminus and A59P in the connecting ␣-helix, our results confirm earlier results obtained by using the phage T4 gene 60-based system to monitor bypassing. The way rplI mutations suppress various frameshift mutations suggests that bypassing of many codons from several takeoff and landing sites occurred instead of a specific frameshift forward at overlapping codons.

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“…The binding site of the L9 protein has been localized to the domain V region of the 23 S rRNA (Adams�i et al, 1996). In addition, L9 has been implicated as a participant in a recoding event �nown as "ribosomal hopping", where L9 mutations have been associated with the ability of ribosomes to bypass a 50-nucleotide region within the coding region of the bacteriophage T4 gene 60 mRNA (Herbst et al, 1994;Adamski et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

cDNA, genomic sequence cloning and overexpression of ribosomal protein gene L9 (rpL9) of the giant panda (Ailuropoda melanoleuca)

Hou

Hou²,

Wu³

et al. 2011

Genet. Mol. Res.

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ABSTRACT. The ribosomal protein L9 (RPL9), a component of the large subunit of the ribosome, has an unusual structure, comprising two compact globular domains connected by an α-helix; it interacts with 23 S rRNA. To obtain information about rpL9 of Ailuropoda melanoleuca (the giant panda), we designed primers based on the �nown mam-, we designed primers based on the �nown mamwe designed primers based on the �nown mammalian nucleotide sequence. RT-PCR and PCR strategies were employed to isolate cDNA and the rpL9 gene from A. melanoleuca; these were sequenced and analyzed. We overexpressed cDNA of the rpL9 gene in Escherichia coli BL21. The cloned cDNA fragment was 627 bp in length, containing an open reading frame of 579 bp. The deduced protein is composed of 192 amino acids, with an estimated molecular mass of 21.86 �Da and an isoelectric point of 10.36. The length of the genomic sequence is 3807 bp, including six exons and five introns. Based on alignment analysis, rpL9 has high similarity among species; we found 85% agreement of DNA and amino acid sequences with the other species that have been analyzed. Based on topology predictions, there are two N-glycosylation sites, five protein kinase C phosphorylation sites, one (2011) cDNA, genomic sequence and overexpression of the rpL9 gene casein �inase II phosphorylation site, two tyrosine �inase phosphorylation sites, three N-myristoylation sites, one amidation site, and one ribosomal protein L6 signature 2 in the L9 protein of A. melanoleuca. The rpL9 gene can be readily expressed in E. coli; it fuses with the N-terminal GST-tagged protein, giving rise to the accumulation of an expected 26.51-�Da polypeptide, which is in good agreement with the predicted molecular weight. This expression product could be used for purification and further study of its function.

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Ribosomal Protein L9 Interactions with 23 S rRNA: The Use of a Translational Bypass Assay to Study the Effect of Amino Acid Substitutions

Cited by 45 publications

References 27 publications

Crippling the Essential GTPase Der Causes Dependence on Ribosomal Protein L9

Crippling the Essential GTPase Der Causes Dependence on Ribosomal Protein L9

Alterations in the Two Globular Domains or in the Connecting α-Helix of Bacterial Ribosomal Protein L9 Induces +1 Frameshifts

cDNA, genomic sequence cloning and overexpression of ribosomal protein gene L9 (rpL9) of the giant panda (Ailuropoda melanoleuca)

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