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Title: Recognition of Ribosomal Protein L11 by the Protein Trimethyltransferase PrmA

Abstract

Bacterial ribosomal protein L11 is post-translationally trimethylated at multiple residues by a single methyltransferase, PrmA. Here, we describe four structures of PrmA from the extreme thermophile Thermus thermophilus. Two apo-PrmA structures at 1.59 and 2.3 {angstrom} resolution and a third with bound cofactor S-adenosyl-L-methionine at 1.75 {angstrom} each exhibit distinct relative positions of the substrate recognition and catalytic domains, revealing how PrmA can position the L11 substrate for multiple, consecutive side-chain methylation reactions. The fourth structure, the PrmA-L11 enzyme-substrate complex at 2.4 {angstrom} resolution, illustrates the highly specific interaction of the N-terminal domain with its substrate and places Lys39 in the PrmA active site. The presence of a unique flexible loop in the cofactor-binding site suggests how exchange of AdoMet with the reaction product S-adenosyl-L-homocysteine can occur without necessitating the dissociation of PrmA from L11. Finally, the mode of interaction of PrmA with L11 explains its observed preference for L11 as substrate before its assembly into the 50S ribosomal subunit.

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
929859
Report Number(s):
BNL-80425-2008-JA
Journal ID: ISSN 0261-4189; EMJODG; TRN: US200822%%1052
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: EMBO Journal; Journal Volume: 26
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BACTERIA; CELL CONSTITUENTS; DISSOCIATION; ENZYMES; INTERACTIONS; METHYLATION; PROTEINS; RESIDUES; SUBSTRATES; national synchrotron light source

Citation Formats

Demirci,H., Gregory, S., Dahlberg, A., and Jogl, G. Recognition of Ribosomal Protein L11 by the Protein Trimethyltransferase PrmA. United States: N. p., 2007. Web. doi:10.1038/sj.emboj.7601508.
Demirci,H., Gregory, S., Dahlberg, A., & Jogl, G. Recognition of Ribosomal Protein L11 by the Protein Trimethyltransferase PrmA. United States. doi:10.1038/sj.emboj.7601508.
Demirci,H., Gregory, S., Dahlberg, A., and Jogl, G. Mon . "Recognition of Ribosomal Protein L11 by the Protein Trimethyltransferase PrmA". United States. doi:10.1038/sj.emboj.7601508.
@article{osti_929859,
title = {Recognition of Ribosomal Protein L11 by the Protein Trimethyltransferase PrmA},
author = {Demirci,H. and Gregory, S. and Dahlberg, A. and Jogl, G.},
abstractNote = {Bacterial ribosomal protein L11 is post-translationally trimethylated at multiple residues by a single methyltransferase, PrmA. Here, we describe four structures of PrmA from the extreme thermophile Thermus thermophilus. Two apo-PrmA structures at 1.59 and 2.3 {angstrom} resolution and a third with bound cofactor S-adenosyl-L-methionine at 1.75 {angstrom} each exhibit distinct relative positions of the substrate recognition and catalytic domains, revealing how PrmA can position the L11 substrate for multiple, consecutive side-chain methylation reactions. The fourth structure, the PrmA-L11 enzyme-substrate complex at 2.4 {angstrom} resolution, illustrates the highly specific interaction of the N-terminal domain with its substrate and places Lys39 in the PrmA active site. The presence of a unique flexible loop in the cofactor-binding site suggests how exchange of AdoMet with the reaction product S-adenosyl-L-homocysteine can occur without necessitating the dissociation of PrmA from L11. Finally, the mode of interaction of PrmA with L11 explains its observed preference for L11 as substrate before its assembly into the 50S ribosomal subunit.},
doi = {10.1038/sj.emboj.7601508},
journal = {EMBO Journal},
number = ,
volume = 26,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • To clone the RPL11 cDNA, we used a polymerase chain reaction (PCR) with the single-stranded cDNA synthesized on the total placentary poly(A){sup +}mRNA with the use of primer M245 containing a 3{prime}-terminal oligo(dT)-tract, the 5{prime}terminal hexadecanucleotide sequence of the M13 universal primer, and a NotiI restriction site between them. On the basis of the known sequence of the 5{prime}-end of the human ribosomal protein L11 mRNA, we chose two partially overlapping deoxyribooligonucleotides as 5{prime}-terminal primers in the amplification of the RPL11 cDNA. A pair of partially overlapping oligonucleotides complementary to the oligo(dT)-containing primer were used as 3{prime}-terminal primers.
  • Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal a-amino group and the -amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal a-amino group in the active site in a trimethylated postcatalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 andmore » in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA.« less
  • No abstract prepared.
  • Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly,more » cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.« less