Iodination of Escherichia coli ribosomal protein L18 abolishes its 5 S RNA binding activity

ABSTRACT Amino acid-deprived rplK (previously known asrelC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative ofrplK encoding L11 lacking the N-terminal 36 amino acids (designated ′L11) was constructed. Bacteria overexpressing ′L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed ′L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.

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Structural behavior of RNA-binding proteins in the free state and in complex with RNA: Escherichia coli ribosomal protein L25 and 5S rRNA

Molecular Biology ◽

10.1134/s0026893308040171 ◽

2008 ◽

Vol 42 (4) ◽

pp. 609-615

Author(s):

Yu. N. Chirgadze

Keyword(s):

Escherichia Coli ◽

Ribosomal Protein ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

5S Rrna ◽

Free State ◽

Structural Behavior

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Characterization of an RNA “binding site” for a specific ribosomal protein of Escherichia coli

MGG Molecular & General Genetics ◽

10.1007/bf00268740 ◽

1972 ◽

Vol 114 (1) ◽

pp. 1-8 ◽

Cited By ~ 53

Author(s):

H. W. Schaup ◽

M. Sogin ◽

C. Woese ◽

C. G. Kurland

Keyword(s):

Escherichia Coli ◽

Ribosomal Protein ◽

Binding Site ◽

Rna Binding

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The rubella virus RNA binding activity of human calreticulin is localized to the N-terminal domain.

Journal of Virology ◽

10.1128/jvi.69.6.3848-3851.1995 ◽

1995 ◽

Vol 69 (6) ◽

pp. 3848-3851 ◽

Cited By ~ 23

Author(s):

C D Atreya ◽

N K Singh ◽

H L Nakhasi

Keyword(s):

Rubella Virus ◽

Rna Binding ◽

Binding Activity ◽

Terminal Domain ◽

Virus Rna

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Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4862-4869.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4862-4869 ◽

Cited By ~ 39

Author(s):

Jörg F. Rippmann ◽

Michaela Klein ◽

Christian Hoischen ◽

Bodo Brocks ◽

Wolfgang J. Rettig ◽

...

Keyword(s):

Escherichia Coli ◽

Proteus Mirabilis ◽

Binding Activity ◽

Host Cells ◽

Heterologous Proteins ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Active Product ◽

Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

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