Alteration in the Acetylation Level of Ribosomal Protein L12 During Growth Cycle of
            <i>Escherichia coli</i>

Ramagopal, Subbanaidu; Subramanian, Alap R.

doi:10.1073/pnas.71.5.2136

Cited by 60 publications

(33 citation statements)

References 28 publications

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“…3), consistent with the presence of four copies/50 S subunit, as has been previously deduced for plastid 70 S ribosome (17). In E. coli, this protein exists in two forms: L12, with free N terminus and L7, ␣-N-acetylated form, the sum of the two forms constituting four copies/50 S ribosomal subunit (51), and the ratio of the two forms altering during the bacterial growth cycle (52). The N terminus of plastid L12 is essentially unmodified (Fig.…”

Section: Post-translational Processing: Plastid 50 S Rps Encoded In Tmentioning

confidence: 51%

The Plastid Ribosomal Proteins

Yamaguchi

Subramanian

2000

Journal of Biological Chemistry

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199

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We have completed identification of all the ribosomal proteins (RPs) in spinach plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using twodimensional electrophoresis, electroblotting/protein sequencing, high performance liquid chromatography purification, polymerase chain reaction-based screening of cDNA library/nucleotide sequencing, and mass spectrometry (reversed-phase HPLC coupled to electrospray ionization mass spectrometry and electrospray ionization mass spectrometry). Spinach plastid 50 S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and two are plastid-specific RPs The plastid (chloroplast) ribosome is a plant-specific, organelle ribosome that produces proteins encoded by the plastid genome. Plastid ribosomes are responsible for the synthesis of huge amounts of biomass, since the large subunit of ribulose 1,5-bisphophate carboxylase/oxygenase (a most abundant protein in the biosphere) is synthesized in plastids. Plastid ribosomes are very similar to the eubacterial 70 S-type ribosome, in composition and general mode of function (1-4). The rRNAs and most of the characterized ribosomal proteins (RPs) 1 in plastid ribosomes also bear close resemblance to the corresponding components so far identified in cyanobacteria, a correlation supporting the importance of endosymbiotic theory in plastid evolution (5).The Escherichia coli ribosome, the most well studied of the eubacterial ribosomes (6), is composed of 21 RPs in the 30 S subunit and 33 RPs in the 50 S subunit. Two more possible E. coli RPs have been suggested: protein Y, the product of E. coli yfia gene (7), bearing a distant sequence homology to a chloroplast-specific RP (PSRP-1), and a protein designated S22 (8). Post-translational modifications are found in many E. coli RPs, although a modification in L16 (Arg 81 ) remains yet to be characterized (see "Results" for plastid L16). We have recently identified all the RPs in spinach plastid 30 S ribosomal subunit, including all its PSRPs and many post-translational modifications (83). The number of RPs in plastid 50 S subunits has so far only been estimated (ϳ35-39; reviewed in Ref.2) and has not been determined.Although the constituents of plastid translational machinery in general are similar to those of E. coli, the genes are distributed in two genome compartments: the plastid and the nucleus. The rRNA and tRNA genes are located in the plastid genome, whereas the genes for processing/modification enzymes, aminoacyl tRNA synthetases, and 60% of the RPs are located in the nuclear genome (1-4). The plastid translation system also differs from the eubacterial system in other significant ways, e.g. chloroplast mRNA is often edited (9); about 60% of chloroplast mRNAs lack canonical ribosome-binding sites found in E. coli mRNAs (10); mRNA levels in chloroplasts remain relatively unchanged through dark/light transitions, whereas protein

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Section: Post-translational Processing: Plastid 50 S Rps Encoded In Tmentioning

confidence: 51%

The Plastid Ribosomal Proteins

Yamaguchi

Subramanian

2000

Journal of Biological Chemistry

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199

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“…The ratio of L7 to L12 is known to change throughout the growth phase (8)(9)(10) and has been linked to variation in the stability of the stalk complex via hydrogen exchange mass spectrometry (MS) experiments (11). L12 CTD and NTD are connected via a flexible hinge region providing mobility to the highly structured CTD (12,13).…”

mentioning

confidence: 99%

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

et al. 2012

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The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. Based on kinetic analysis we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.Protein biosynthesis is carried out by the ribosomal machinery and requires interaction of several translation factors with the ribosomal stalk complex. In bacteria the base of the stalk region, interacting with the rRNA of the 50S large ribosomal subunit, is composed of adjacent proteins L11 and L10 (1). The C-terminal domain (CTD) of L10 is a long and mobile helix which interacts with two dimers of protein L12 (2). Three pairs of L12 are observed in thermophilic bacteria and archaea (3-6) and L12 is the only protein present as multiple copies on the ribosome. The N-terminal domain (NTD) of L12 is responsible for dimerization and for anchoring of the protein to the ribosome (7). In Escherichia coli (E. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts coli), L12 is partially N-acetylated to form protein L7 (8). The ratio of L7 to L12 is known to change throughout the growth phase (8-10) and has been linked to variation in the stability of the stalk complex via hydrogen exchange mass spectrometry (MS) experiments (11). L12 CTD and NTD are connected via a flexible hinge region providing mobility to the highly structured CTD (12, 13). These highly mobile proteins have eluded high resolution X-ray analysis for many years. Only recently the atomic structure of a truncated stalk complex of a thermophilic bacterium revealed the binding mode of the NTD of L12 proteins to L10 (4). The tight antiparallel binding of the L12 NTDs is similar to that observed by NMR for the E. coli L7/L12 dimer in solution (14). The mobility and dynamics o...

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“…All of the model building and refinement were carried out using the programs O (29) and CNS (30). Details of the refined structures are reported in In E. coli and Salmonella, the ratio of L12/L7 was found to vary with growth where the proportion as L12 was highest during logarithmic growth (12)(13)(14). To generate L12 substrate, cells expressing L12…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

“…This is due to the interaction of the C-terminal domains of L12/L7 with both elongation factors EF-Tu and EF-G and the promotion of GTP hydrolysis by these two elongation factors (10, 11). Finally, whereas S18 and S5 are always found in their N ␣ -acetylated form, the relative proportions of L12 and L7 are correlated with the rate of growth and the cell cycle (12)(13)(14).Although cotranslational N ␣ -acetylation appears to be critical to the function and stability of numerous eukaryotic proteins (15), no known function has been ascribed to the acetylation of prokaryotic ribosomal proteins. Knock-out mutants lacking a functional RimL, RimI, or RimJ exhibit no distinguishable phenotype compared with wild type (16 -18), and the L12/L7 ratio has not been found to affect ribosomal function (19 -21).…”

mentioning

confidence: 99%

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A Novel Dimeric Structure of the RimL Nα-acetyltransferase from Salmonella typhimurium

Vetting

Carvalho

Roderick

et al. 2005

Journal of Biological Chemistry

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RimL is responsible for converting the prokaryotic ribosomal protein from L12 to L7 by acetylation of its N-terminal amino group. We demonstrate that purified RimL is capable of posttranslationally acetylating L12, exhibiting a V max of 21 min ؊1 . We have also determined the apostructure of RimL from Salmonella typhimurium and its complex with coenzyme A, revealing a homodimeric oligomer with structural similarity to other Gcn5-related N-acetyltransferase superfamily members. A large central trough located at the dimer interface provides sufficient room to bind both L12 N-terminal helices. Structural and biochemical analysis indicates that RimL proceeds by single-step transfer rather than a covalent-enzyme intermediate. This is the first structure of a Gcn5-related N-acetyltransferase family member with demonstrated activity toward a protein N ␣ -amino group and is a first step toward understanding the molecular basis for N ␣ acetylation and its function in cellular regulation.The posttranslational acetylation of N ␣ -protein termini is a common occurrence in eukaryotic proteins where between 50 and 90% of proteins are N ␣ -acetylated (1). However, in prokaryotes, N ␣ -protein acetylation is limited to four known examples. The early secreted antigenic target (ESAT-6) of Mycobacterium tuberculosis is N ␣ -acetylated, and its acetylation has been shown to alter its interaction with the 10-kDa culture filtrate protein (CFP10) (2). The three other examples are the prokaryotic ribosomal proteins S18, S5, and L12, which are N ␣ -acetylated by the Rim proteins, RimI, RimJ, and RimL, respectively (3, 4). L12 and its N ␣ -acetylated derivative, L7, have garnered the most interest because of its interesting physical and biological properties. L12 lacks tryptophan, tyrosine, cysteine, and histidine residues and is the most acidic Escherichia coli ribosomal protein (5). The dimeric L12/L7 complex is the only protein found in more than one copy in the ribosome (6) and is the only ribosomal protein that has no direct contact with rRNA, instead interacting indirectly via formation of a complex with the L10 protein (7). In addition, the efficiency of protein translation is correlated with the number of L12/L7 dimers bound with maximum efficiency occurring at a ratio of two dimers per ribosome (8,9). This is due to the interaction of the C-terminal domains of L12/L7 with both elongation factors EF-Tu and EF-G and the promotion of GTP hydrolysis by these two elongation factors (10, 11). Finally, whereas S18 and S5 are always found in their N ␣ -acetylated form, the relative proportions of L12 and L7 are correlated with the rate of growth and the cell cycle (12)(13)(14).Although cotranslational N ␣ -acetylation appears to be critical to the function and stability of numerous eukaryotic proteins (15), no known function has been ascribed to the acetylation of prokaryotic ribosomal proteins. Knock-out mutants lacking a functional RimL, RimI, or RimJ exhibit no distinguishable phenotype compared with wild type (16 -18), and the L12/L...

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Alteration in the Acetylation Level of Ribosomal Protein L12 During Growth Cycle of Escherichia coli

Cited by 60 publications

References 28 publications

The Plastid Ribosomal Proteins

The Plastid Ribosomal Proteins

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

A Novel Dimeric Structure of the RimL Nα-acetyltransferase from Salmonella typhimurium

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