Differential Effects of ReplacingEscherichiacoliRibosomal Protein L27 with Its Homologue fromAquifexaeolicus

Maguire, Bruce A.; Manuilov, Anton V.; Zimmermann, Robert A.

doi:10.1128/jb.183.22.6565-6572.2001

Cited by 5 publications

(4 citation statements)

References 49 publications

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“…No interactions between proteins and substrates at or near the PTC were observed in structures of the 50S subunit from the archaeon Haloarcula marismortui 1 , 28 . However archaea do not contain a homolog of L27, which is known to cross-link to the 3′ ends of the tRNA substrates in bacteria 7 , 29 . Further, the relevant regions of protein L10e, an archaeal homolog of L16, were disordered in these 50S structures, possibly because they lacked a full-length tRNA substrate.…”

Section: Discussionmentioning

confidence: 99%

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Voorhees

Weixlbaumer

Loakes

et al. 2009

Nat Struct Mol Biol

339

409

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Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. Additionally, though structures of the 50S subunit found no ordered proteins at the PTC, biochemical evidence suggests specific proteins are capable of interacting with the 3′ ends of the tRNA ligands. Here we present structures at 3.5 Å and 3.55 Å resolution of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. Additionally they reveal interactions between ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer.

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Section: Discussionmentioning

confidence: 99%

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Voorhees

Weixlbaumer

Loakes

et al. 2009

Nat Struct Mol Biol

339

409

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“…To construct this plasmid, the rpmA gene obtained from plasmid pETrpmA was amplified using PR3 and PR4 and cloned into pET-23a (see Fig. 2A) (20). Plasmid pETrpmA-At-2 was a derivative of pETrpmA-At-1 into which the ssrA gene with its flanking regions (133 bp upstream and 157 bp downstream) was inserted.…”

Section: Methodsmentioning

confidence: 99%

Contributions of Pseudoknots and Protein SmpB to the Structure and Function of tmRNA in trans-Translation

Wower

Zwieb

Wower

2004

Journal of Biological Chemistry

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Bacteria contain transfer-messenger RNA (tmRNA), a molecule that during trans-translation tags incompletely translated proteins with a small peptide to signal the proteolytic destruction of defective polypeptides. TmRNA is composed of tRNA-and mRNA-like domains connected by several pseudoknots. Using truncated ribosomal protein L27 as a reporter for tagging in vitro and in vivo, we have developed exceptionally sensitive assays to study the role of Escherichia coli tmRNA in trans-translation. Site-directed mutagenesis experiments showed that pseudoknot 2 and the abutting helix 5 were particularly important for the binding of ribosomal protein S1 to tmRNA. Pseudoknot 4 not only facilitated tmRNA maturation but also promoted tagging. In addition, the three pseudoknots (pk2 to pk4) were shown to play a significant role in the proper folding of the tRNA-like domain. Protein SmpB enhanced tmRNA processing, suggesting a new role for SmpB in transtranslation. Taken together, these results provide unanticipated insights into the functions of the pseudoknots and protein SmpB during tmRNA folding, maturation, and protein synthesis.An interruption of the elongation step of protein synthesis results in the production of truncated proteins and leaves ribosomes stalled at the 3Ј end of mRNA templates lacking a stop codon(s). To remove the defective polypeptides and recycle the ribosomes, bacteria have developed trans-translation, a quality control mechanism that tags the C termini of defective proteins with a short peptide recognized by housekeeping proteases. This peptide tag is encoded by a short open reading frame present in a small stable RNA molecule called 10Sa or transfermessenger RNA (tmRNA).1 It has been shown that in addition to protein tagging, tmRNA facilitates the recycling of ribosomes by providing missing stop codons (1, 2).Structure probing of the Escherichia coli tmRNA and sequence comparisons demonstrated the presence of three domains (Fig. 1A) (3-5). The 3Ј and 5Ј termini of the tmRNA form the tRNA-like domain (TLD) with a significantly reduced D arm. The resume codon and stop codon(s) demarcate the open reading frame in the mRNA-like domain (MLD). The TLD and the MLD are connected by a pseudoknot-rich domain consisting of four pseudoknots (pk1 to pk4) in most tmRNAs.The three-dimensional model of E. coli tmRNA suggests a structure in which the TLD is connected to the circularly arranged MLD and pseudoknots through coaxially stacked helices (6). Recently, the entry of tmRNA into a stalled E. coli ribosome has been visualized by cryo-electron microscopy (7). At this particular step of trans-translation, the TLD, pk1, and the MLD contact the ribosome, whereas the pk2 to pk4 segment forms an arc that remains outside the ribosome.Three proteins facilitate binding of tmRNA to the ribosome. Elongation factor Tu forms a ternary complex with GTP and aminoacyl-tmRNA, as in regular protein synthesis (8, 9). Protein SmpB binds to the ternary complex in vitro as well as to stalled ribosomes in vivo (10 -12). Ribosomal p...

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“…Extraction of ribosomal proteins from 70S ribosomes and 1D and 2D PAGE were performed as described (Maguire et al, 2001), except that the latter employed the pH 8.6 first-dimension gel of Madjar et al (1979). Images of stained gels were captured by using a Kodak DC290 digital camera and the Kodak ID LE program.…”

Section: Electrophoresis Of Ribosomal Proteinsmentioning

confidence: 99%

A Protein Component at the Heart of an RNA Machine: The Importance of Protein L27 for the Function of the Bacterial Ribosome

et al. 2005

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Deletion of the gene for protein L27 from the E. coli chromosome results in severe defects in cell growth. This deficiency is corrected by the expression of wild-type (wt) protein L27 from a plasmid. Examination of strains expressing L27 variants truncated at the N terminus reveals that the absence of as few as three amino acids leads to a decrease in growth rate, an impairment in peptidyl transferase activity, and a sharp decline in the labeling of L27 from the 3' end of a photoreactive tRNA at the ribosomal P site. These findings suggest that the flexible N-terminal sequence of L27, which protrudes onto the interface of the bacterial 50S subunit, can reach the peptidyl transferase active site and contribute to its function, possibly by helping to correctly position tRNA substrates at the catalytic site.

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Differential Effects of ReplacingEscherichiacoliRibosomal Protein L27 with Its Homologue fromAquifexaeolicus

Cited by 5 publications

References 49 publications

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Contributions of Pseudoknots and Protein SmpB to the Structure and Function of tmRNA in trans-Translation

A Protein Component at the Heart of an RNA Machine: The Importance of Protein L27 for the Function of the Bacterial Ribosome

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