Restriction Site-Free Insertion of PCR Products Directionally into Vectors

Chen, G.J.; Qiu, NaHong; Karrer, Catherine; Caspers, Patrick; Page, Malcolm G. P.

doi:10.2144/00283st08

Cited by 58 publications

(56 citation statements)

References 9 publications

(8 reference statements)

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“…The DNA fragment encoding Thermus thermophilus ClpB ΔN (amino acids 143-854) was generated by PCR using Thermus thermophilus ClpB (7) as a template and subsequently cloned into a modified pET28b vector, with the thrombin cleavage site replaced by a six-histidine tag followed by a tobacco etch virus (TEV) protease cleavage site. The NTD domain of ClpB (amino acids 1-142) was prepared by a ligation-free cloning method (40,41) with residues 1-142 of ClpB PCR amplified with appropriate primers from a full-length ClpB vector and inserted into a pTWIN1 vector (NEB) immediately in front of the Mxe GyrA intein (an entire chitin binding domain, Ssp DnaB intein, and the MCS cassette was replaced by residues 1-142 of ClpB). An uncleavable hexahistidine tag was added at the N terminus of the construct by ligation of a duplex oligonucleotide containing five his codons (flanked by NdeI overhangs on both sides) into a NdeI-cleaved NTD plasmid.…”

Section: Methodsmentioning

confidence: 99%

ClpB N-terminal domain plays a regulatory role in protein disaggregation

Rosenzweig

Farber

Vėlyvis

et al. 2015

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

114

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ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/ Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.T he heat shock protein ClpB (Escherichia coli) or Hsp100 (eukaryotes) is the main protein disaggregase in bacteria, yeast, plants, and mitochondria of all eukaryotic cells, and it is essential for cell survival during severe stress (1-4). Recovery of functional proteins from aggregates by ClpB requires the synergistic interaction with a second molecular chaperone, DnaK (1). Through its cochaperone, DnaJ, DnaK initially binds to the aggregates, leading to the exposure of peptide segments that can be recognized by ClpB (5, 6). DnaK then recruits ClpB to the site of aggregation through direct physical interaction (7, 8), transferring the aggregate to ClpB. Using the energy derived from ATP hydrolysis, ClpB unravels the aggregate by threading single polypeptide chains, one at a time, through the central pore of its hexameric ring (9). Once released from the aggregate, the unfolded polypeptides can either refold spontaneously or fold with the help of additional cellular chaperones.Like other Hsp100 proteins, ClpB forms a hexameric ring, with each protomer comprising an N-terminal domain (NTD) and two nucleotide binding domains (NBD1 and NBD2) separated by a unique regulatory coil-coil domain (10) essential for DnaK binding (7, 11) ( Fig. 1 A and B). Both NBDs contain Walk...

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Section: Methodsmentioning

confidence: 99%

ClpB N-terminal domain plays a regulatory role in protein disaggregation

Rosenzweig

Farber

Vėlyvis

et al. 2015

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

114

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“…The components indispensable to the cell-free protein synthesis, including ribosomes, IF1, IF2, IF3, EF-G, EF-Tu, EF-Ts, RF1, RF3, RRF, 20 aminoacyl-tRNA synthetase, methionyl-tRNA transformylase, T7 RNA polymerase and nucleoside-diphosphate kinase (NDK), are puri¢ed according to the previous report [10]. The gene encoding tmRNA was ampli¢ed by PCR from the E. coli genome and cloned into vector pET17b (Novagen) using a restriction site-free method described by Chen et al [11] with a slight modi¢cation. The obtained plasmid, containing a T7 promoter directly upstream of the ssrA gene, was transformed into E. coli JM109/DE3 strain.…”

Section: Introductionmentioning

confidence: 99%

The role of SmpB protein in trans‐translation

Shimizu

Ueda

2002

FEBS Letters

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The function of SmpB protein in the trans-translation system was evaluated using the well-defined cell-free translation system consisting of purified ribosome, alanyl-tRNA synthetase and elongation factors. The analysis showed that SmpB protein enhances alanine-accepting activity of tmRNA and that SmpB protein and tmRNA are sufficient to complete the transtranslation process in the presence of translational components. Moreover, SmpB is indispensable in the addition of tag-peptide onto ribosomes by tmRNA. In particular, the A-site binding of tmRNA is inhibited in the absence of SmpB. ß

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“…Cloning was accomplished using a protocol similar to the QuikChange method (Agilent) as described previously (68). Briefly, the QuikChange method allows genes to be inserted into vectors via linear amplification using PfuUltra II Fusion HF polymerase (Agilent), avoiding the introduction of cloning artifacts and resulting in faster preparation of constructs (70,71). The sequences of the constructs and coiled-coil registry are detailed in Table 5.…”

Section: Methodsmentioning

confidence: 99%

Family-specific Kinesin Structures Reveal Neck-linker Length Based on Initiation of the Coiled-coil

Phillips

Peter

Gilbert

et al. 2016

Journal of Biological Chemistry

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and -7 generate processive hand-over-hand 8-nm steps to transport intracellular cargoes toward the microtubule plus end. This processive motility requires gating mechanisms to coordinate the mechanochemical cycles of the two motor heads to sustain the processive run. A key structural element believed to regulate the degree of processivity is the necklinker, a short peptide of 12-18 residues, which connects the motor domain to its coiled-coil stalk. Although a shorter necklinker has been correlated with longer run lengths, the structural data to support this hypothesis have been lacking. To test this hypothesis, seven kinesin structures were determined by x-ray crystallography. Each included the neck-linker motif, followed by helix ␣7 that constitutes the start of the coiled-coil stalk. In the majority of the structures, the neck-linker length differed from predictions because helix ␣7, which initiates the coiled-coil, started earlier in the sequence than predicted. A further examination of structures in the Protein Data Bank reveals that there is a great disparity between the predicted and observed starting residues. This suggests that an accurate prediction of the start of a coiled-coil is currently difficult to achieve. These results are significant because they now exclude simple comparisons between members of the kinesin superfamily and add a further layer of complexity when interpreting the results of mutagenesis or protein fusion. They also re-emphasize the need to consider factors beyond the kinesin neck-linker motif when attempting to understand how inter-head communication is tuned to achieve the degree of processivity required for cellular function.

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Restriction Site-Free Insertion of PCR Products Directionally into Vectors

Cited by 58 publications

References 9 publications

ClpB N-terminal domain plays a regulatory role in protein disaggregation

ClpB N-terminal domain plays a regulatory role in protein disaggregation

The role of SmpB protein in trans‐translation

Family-specific Kinesin Structures Reveal Neck-linker Length Based on Initiation of the Coiled-coil

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