Crystal structure analysis of the translation factor RF3 (release factor 3)

Kihira, Kiyohito; Shimizu, Yoshihiro; Shomura, Yasuhito; Shibata, Naoki; Kitamura, Masaya; Nakagawa, Atsushi; Ueda, Takuya; Ochi, Kozo; Higuchi, Yoshiki

doi:10.1016/j.febslet.2012.08.029

Cited by 28 publications

(32 citation statements)

References 14 publications

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“…In addition, examining the surface potential in RF3 from D . vulgaris (Figure 6G) (Kihira et al, 2012) we find a similar positive surface potential in the same position as we observed for RF3 from E . coli .…”

Section: Resultssupporting

confidence: 87%

Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1

Pallesen

Hashem

Korkmaz

et al. 2013

eLife

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Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.DOI: http://dx.doi.org/10.7554/eLife.00411.001

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Section: Resultssupporting

confidence: 87%

Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1

Pallesen

Hashem

Korkmaz

et al. 2013

eLife

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“…High-resolution crystal structures provide insights into these interactions. So far, several (p)ppGpp-bound structures have been solved, including E. coli RNA polymerase (PDB: 4JK1 and 4JKR, ~4 Å resolution) (Mechold et al, 2013; Zuo et al, 2013), lysine decarboxylase (PDB: 3N75, 2.0 Å) (Kanjee et al, 2011), adenylosuccinate synthetase (PDB: 1CH8, 2.5 Å) (Hou et al, 1999), Desulfovibrio vulgaris translation release factor 3 (PDB: 3VR1, 3.0 Å) (Kihira et al, 2012), S. aureus primase (PDB: 4EDT, 2.01Å) (Rymer et al, 2012), Aquifex aeolicus PPX/GPPA phosphatase (PDB: 2J4R, 2.71 Å) (Kristensen et al, 2008), and B. subtilis Obg (PDB: 1LNZ, 2.6 Å) (Buglino et al, 2002). Here, we described the 1.67 Å resolution crystal structure of pppGpp-bound S. aureus GMK, which is the highest resolution achieved for a (p)ppGpp-bound structure to date.…”

Section: Discussionmentioning

confidence: 99%

Molecular Mechanism and Evolution of Guanylate Kinase Regulation by (p)ppGpp

et al. 2015

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SUMMARY The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK) revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.

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“…ppGpp can also bind to and affect the activities of a large number of other proteins in addition to RNAP (1,2), thereby playing roles in processes as diverse as DNA replication (8,9), translation (10)(11)(12), nucleotide metabolism (2,(11)(12)(13)(14), ribosome biogenesis (11,12,15,16), amino acid decarboxylation (17), polyphosphate metabolism (18), and ppGpp metabolism itself (11,19,20). Our studies were designed to distinguish the effects of ppGpp on gene expression resulting from its binding to RNAP versus other cellular targets.…”

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confidence: 99%

Genome-wide effects on Escherichia coli transcription from ppGpp binding to its two sites on RNA polymerase

Sanchez-Vazquez

Dewey

Kitten

et al. 2019

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

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The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) inEscherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.

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Crystal structure analysis of the translation factor RF3 (release factor 3)

Cited by 28 publications

References 14 publications

Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1

Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1

Molecular Mechanism and Evolution of Guanylate Kinase Regulation by (p)ppGpp

Genome-wide effects on Escherichia coli transcription from ppGpp binding to its two sites on RNA polymerase

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