Protein Unfolding and Degradation by the CLP Family of Proteases

ATP-dependent proteases play a major role in regulatory protein degradation in both prokaryotic and eukaryotic cells. ATP-dependent proteases in E. coli fall into two classes. The first class requires the interaction of structurally separate proteases with an ATPase, whereas in the second class both the protease and ATPase are formed from regions of the same polypeptide chain. We have studied the structure of several of these protein degrading complexes in E. coli and have found a remarkable similarity in the architecture of these macromolecular assemblies.The prototypical protease of the first class has as its proteolytic component ClpP, a 14 subunit (MW 21,500) complex arranged as two lOnm-diameter stacked rings of seven subunits each. ClpP can interact with either one of two ATPases, ClpA or ClpX, each with unique substrate specificity. ClpA has two ATP-binding sites per subunit (MW 84,000), and its subunits are arranged as a 13nm (diameter) hexameric ring (MW -500,000).

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Polypeptide Chain Initiation and the Characterization of Ribosomal Binding Sites in E. coli

Cold Spring Harbor Symposia on Quantitative Biology ◽

10.1101/sqb.1966.031.01.038 ◽

1966 ◽

Vol 31 (0) ◽

pp. 289-295 ◽

Cited By ~ 11

Author(s):

M. S. Bretscher

Keyword(s):

Binding Sites ◽

Polypeptide Chain ◽

Chain Initiation ◽

E Coli

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High-resolution gold labeling

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147491 ◽

1993 ◽

Vol 51 ◽

pp. 330-331

Author(s):

James F. Hainfeld ◽

Frederic R. Furuya ◽

Kyra Carbone ◽

Martha Simon ◽

Beth Lin ◽

...

Keyword(s):

Dihydrofolate Reductase ◽

Polypeptide Chain ◽

External Surface ◽

Gold Cluster ◽

Macromolecular Complex ◽

Gold Compound ◽

Central Cavity ◽

Chain Folding ◽

E Coli ◽

Chaperonin Groel

A recently developed 1.4 nm gold cluster has been found to be useful in labeling macromolecular sites to 1-3 nm resolution. The gold compound is organically derivatized to contain a monofunctional arm for covalent linking to biomolecules. This may be used to mark a specific site on a structure, or to first label a component and then reassemble a multicomponent macromolecular complex. Two examples are given here: the chaperonin groEL and ribosomes.Chaperonins are essential oligomeric complexes that mediate nascent polypeptide chain folding to produce active proteins. The E. coli chaperonin, groEL, has two stacked rings with a central hole ∽6 nm in diameter. The protein dihydrofolate reductase (DHFR) is a small protein that has been used in chain folding experiments, and serves as a model substrate for groEL. By labeling the DHFR with gold, its position with respect to the groEL complex can be followed. In particular, it was sought to determine if DHFR refolds on the external surface of the groEL complex, or whether it interacts in the central cavity.

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Global versus Local Regulatory Roles for Lrp-Related Proteins: Haemophilus influenzae as a Case Study

Journal of Bacteriology ◽

10.1128/jb.183.13.4004-4011.2001 ◽

2001 ◽

Vol 183 (13) ◽

pp. 4004-4011 ◽

Cited By ~ 20

Author(s):

Devorah Friedberg ◽

Michael Midkiff ◽

Joseph M. Calvo

Keyword(s):

Amino Acid ◽

Haemophilus Influenzae ◽

Regulatory Protein ◽

Enteric Bacteria ◽

Regulatory Role ◽

Regulatory Function ◽

Ecological Niches ◽

E Coli ◽

Sequence Identity ◽

Related Proteins

ABSTRACT Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, includingasnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated withE. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein fromHaemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having anlrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies withlrp + and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. colicell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function.

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A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽

10.1128/mbio.01105-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 7

Author(s):

Christopher W. Lennon ◽

Kimberly C. Lemmer ◽

Jessica L. Irons ◽

Max I. Sellman ◽

Timothy J. Donohue ◽

...

Keyword(s):

Escherichia Coli ◽

Structure Function ◽

Rhodobacter Sphaeroides ◽

Fatty Acid Content ◽

Regulatory Protein ◽

Growth Conditions ◽

Globular Domain ◽

Content Type ◽

E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

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Genome-Wide Identification of Transcription Start Sites, Promoters and Transcription Factor Binding Sites in E. coli

PLoS ONE ◽

10.1371/journal.pone.0007526 ◽

2009 ◽

Vol 4 (10) ◽

pp. e7526 ◽

Cited By ~ 184

Author(s):

Alfredo Mendoza-Vargas ◽

Leticia Olvera ◽

Maricela Olvera ◽

Ricardo Grande ◽

Leticia Vega-Alvarado ◽

...

Keyword(s):

Transcription Factor ◽

Binding Sites ◽

Transcription Factor Binding Sites ◽

Transcription Factor Binding ◽

Transcription Start ◽

Transcription Start Sites ◽

E Coli ◽

Factor Binding ◽

Genome Wide

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Effects of cations on binding of human choriogonadotropin

Biochemistry and Cell Biology ◽

10.1139/o88-145 ◽

1988 ◽

Vol 66 (12) ◽

pp. 1258-1264

Author(s):

Patrick J. McIlroy

Keyword(s):

Binding Sites ◽

Regulatory Protein ◽

Guanine Nucleotides ◽

Guanine Nucleotide ◽

Human Choriogonadotropin ◽

Receptor Sites ◽

Number Of Binding Sites ◽

Receptor Number ◽

Guanine Nucleotide Regulatory Protein ◽

Low Ionic Strength

The effect of various salts on the binding of human choriogonadotropin to rat luteal membranes has been examined. Increasing salt concentrations had biphasic effects, initially increasing binding, then decreasing it. With NaCl, these effects were on both the affinity and the number of receptor sites. The affinity increased with increasing NaCl concentrations, to a maximum at 40 mM, and then decreased. Above 40 mM NaCl, the number of binding sites increased. NaCl also altered the effects of Mg2+ and guanyl nucleotides. At low ionic strength, Mg2+ was necessary to observe binding. Guanine nucleotides modulated this binding by decreasing the affinity. At 40 mM NaCl, Mg2+ increased receptor number without altering affinity. Guanyl nucleotides modulated this binding by reducing the number of sites to that observed in the absence of Mg2+. At 150 mM NaCl, Mg2+ and guanine nucleotides had no effect. The results suggest the presence of two pools of human choriogonadotropin receptor in rat corpus luteum, one coupled to the guanine nucleotide regulatory protein (Ns) and being Mg2+ dependent and guanine nucleotide sensitive, and the other not coupled to Ns and being Mg2+ independent and guanine nucleotide insensitive.

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