ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle

Crooke, Elliott; Guthrie, Brenda; Lecker, Stewart H.; Lill, Roland; Wickner, William

doi:10.1016/0092-8674(88)90115-8

Cited by 194 publications

(162 citation statements)

References 38 publications

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“…All other chemicals were purchased from Sigma (St Louis, MO, USA). SecA, 38 SecB 39 and proOmpA 40 were purified essentially as described. GST, GST-SecY(Tyr332-Tyr365) and GST-SecY (Phe170-Gly184) were purified with glutathione-Sepharose 4B according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

Sluis¹,

Nouwen²,

Koch³

et al. 2006

Journal of Molecular Biology

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Section: Methodsmentioning

confidence: 99%

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

Sluis¹,

Nouwen²,

Koch³

et al. 2006

Journal of Molecular Biology

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“…It has been demonstrated that TF has a binding affinity to this protein when going from a denaturant like urea to renaturation conditions by either dilution or dialysis [2]. Therefore, stoichiometric complexes of TF and proOmpA could be isolated [2,3]. Using genetically engineered strains of E. coli either over-or underproducing TF, however, the membrane transport of proOmpA was not affected [4].…”

Section: Introductionmentioning

confidence: 99%

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

Stoller¹,

Tradler²,

Rücknagel³

et al. 1996

FEBS Letters

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The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl-prolyl cisltrans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatieally active fragment could be isolated after proteolysis by subtifisin. The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251. The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His-tagged polypeptide was isolated on an Ni 2+-NTA column. Subsequent digestion with subtifisin and anionexchange chromatography yielded a TF fragment encompassing amino acids Gin-148 to Thr-249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 ~1-1 s -l could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrofide FK506.

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“…In contrast, Lep75-RNCs bound TF with low affinity as vacant ribosomes, indicating that TF does not interact with the hydrophobic signal-anchor sequence of Lep. However, given the high concentration of TF in the cell (about 50 mM total 30 ), TF can probably bind to most ribosomes, including non-translating ribosomes and RNCs, even to those RNCs that do not present a TF-binding motif. This raises the question of how the other RPBs gain access to the nascent peptide for N-terminal processing (PDF, MAP) and membrane targeting (SRP).…”

Section: Kinetics Of Tf Interaction With Non-translating Ribosomesmentioning

confidence: 99%

Interplay between trigger factor and other protein biogenesis factors on the ribosome

2014

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Nascent proteins emerging from translating ribosomes in bacteria are screened by a number of ribosome-associated protein biogenesis factors, among them the chaperone trigger factor (TF), the signal recognition particle (SRP) that targets ribosomes synthesizing membrane proteins to the membrane and the modifying enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). Here, we examine the interplay between these factors both kinetically and at equilibrium. TF rapidly scans the ribosomes until it is stabilized on ribosomes presenting TF-specific nascent chains. SRP binding to those complexes is strongly impaired. Thus, TF in effect prevents SRP binding to the majority of ribosomes, except those presenting SRP-specific signal sequences, explaining how the small amount of SRP in the cell can be effective in membrane targeting. PDF and MAP do not interfere with TF or SRP binding to translating ribosomes, indicating that nascent-chain processing can take place before or in parallel with TF or SRP binding.

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ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle

Cited by 194 publications

References 38 publications

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

Interplay between trigger factor and other protein biogenesis factors on the ribosome

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