GTPases IF2 and EF-G bind GDP and the SRL RNA in a mutually exclusive manner

Mitkevich, Vladimir A.; Shyp, Viktoriya; Petrushanko, Irina Yu.; Soosaar, Andres; Atkinson, Gemma C.; Tenson, Tanel; Makarov, Alexander А.; Hauryliuk, Vasili

doi:10.1038/srep00843

Cited by 10 publications

(11 citation statements)

References 59 publications

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“…The thermodynamic parameters of Aβ(1-42) binding to Na,K-ATPase were measured using a MicroCal iTC200 instrument, as described elsewhere 44 45 . Experiments were carried out at 25 °C in 10 мМ imidazole buffer (pH 7.5), containing 130 мМ NaCl, 30 мM KCl, 3 мМ MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function

Petrushanko

Mitkevich

Anashkina

et al. 2016

Sci Rep

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By maintaining the Na+ and K+ transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aβ) at the early stages of Alzheimer’s disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aβ(1-42) forms a tight (Kd of 3 μM), enthalpy-driven equimolar complex with α1β1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aβ(1-42) is localized in the “gap” between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aβ(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aβ(1-42) level. However prolonged increase of Aβ(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

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Section: Methodsmentioning

confidence: 99%

Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function

Petrushanko

Mitkevich

Anashkina

et al. 2016

Sci Rep

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“…However, simple geometric considerations based on available cryoEM reconstitutions indicate that both conformation and position of IF2 on the ribosome must be changed to allow 30S IC -50S association. Contact between IF2 and the GAC (GTPase Activating Center) and the SRL (Sarcin Ricin Loop) of the 50S subunit [ 94 , 101 , 155 – 157 ] triggers a very rapid (30–45 s −1 ) IF2-dependent GTP hydrolysis (Fig. 7 b, Step 8) [ 124 , 132 , 150 ].…”

Section: Formation Of the 30s And 70s Initiation Complexesmentioning

confidence: 99%

Initiation of mRNA translation in bacteria: structural and dynamic aspects

Gualerzi

Pon

2015

Cell. Mol. Life Sci.

142

132

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Initiation of mRNA translation is a major checkpoint for regulating level and fidelity of protein synthesis. Being rate limiting in protein synthesis, translation initiation also represents the target of many post-transcriptional mechanisms regulating gene expression. The process begins with the formation of an unstable 30S pre-initiation complex (30S pre-IC) containing initiation factors (IFs) IF1, IF2 and IF3, the translation initiation region of an mRNA and initiator fMet-tRNA whose codon and anticodon pair in the P-site following a first-order rearrangement of the 30S pre-IC produces a locked 30S initiation complex (30SIC); this is docked by the 50S subunit to form a 70S complex that, following several conformational changes, positional readjustments of its ligands and ejection of the IFs, becomes a 70S initiation complex productive in initiation dipeptide formation. The first EF-G-dependent translocation marks the beginning of the elongation phase of translation. Here, we review structural, mechanistic and dynamical aspects of this process.

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“…As well as being crucial for activation of GTPase activity ( 23 , 24 ), the SRL serves as an affinity point for their association with the ribosome ( 25 ). The isolated SRL rRNA oligonucleotide fragment binds to translational GTPases EF-G and IF2 with μM-range affinity, and complex formation is abolished by GDP ( 26 , 27 ). The SRL is targeted by toxins alpha-sarcin and ricin—the namesakes of this functional element—that inactivate the ribosome by cleaving the phosphodiester bond between G2661–2662 and depurinating A2660, respectively ( 28 , 29 ).…”

Section: Introductionmentioning

confidence: 99%

“…Guided by recent structural insights ( 9–11 ), we have applied a combination of biochemical and microbiological techniques to probe RelA’s molecular mechanism to establish structure–functional relationships. With the exception of one study using an N-terminally tagged construct from the ASKA library ( 38 , 39 ), earlier biochemical studies that were performed with recombinantly produced Escherichia coli RelA relied on C-terminal His 6 -tagging for purification ( 20 , 21 , 26 ). Given the location of RelA’s C-terminus deep inside the ribosomal complex, it is likely that the C-terminal His 6 tag affects the functionality of the protein causing experimental artifacts.…”

Section: Introductionmentioning

confidence: 99%

The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA

Kudrin

Dzhygyr

Ishiguro

et al. 2018

Nucleic Acids Research

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During amino acid starvation the Escherichia coli stringent response factor RelA recognizes deacylated tRNA in the ribosomal A-site. This interaction activates RelA-mediated synthesis of alarmone nucleotides pppGpp and ppGpp, collectively referred to as (p)ppGpp. These two alarmones are synthesized by addition of a pyrophosphate moiety to the 3′ position of the abundant cellular nucleotide GTP and less abundant nucleotide GDP, respectively. Using untagged native RelA we show that allosteric activation of RelA by pppGpp increases the efficiency of GDP conversion to achieve the maximum rate of (p)ppGpp production. Using a panel of ribosomal RNA mutants, we show that the A-site finger structural element of 23S rRNA helix 38 is crucial for RelA binding to the ribosome and consequent activation, and deletion of the element severely compromises (p)ppGpp accumulation in E. coli upon amino acid starvation. Through binding assays and enzymology, we show that E. coli RelA does not form a stable complex with, and is not activated by, deacylated tRNA off the ribosome. This indicates that in the cell, RelA first binds the empty A-site and then recruits tRNA rather than first binding tRNA and then binding the ribosome.

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GTPases IF2 and EF-G bind GDP and the SRL RNA in a mutually exclusive manner

Cited by 10 publications

References 59 publications

Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function

Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function

Initiation of mRNA translation in bacteria: structural and dynamic aspects

The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA

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