Kasugamycin Function in E. coli 30S Ribosomal Subunit: inhibition of translation

Edna Kemboi '16 and Jiayu Chen '17


I. Introduction

Kasugamycin (Ksg) is an antibiotic produced by the bacterium Streptomyces kasugaensis. It has been reported that it can inhibit initiation of translation by blocking initiator transfer RNA binding to the 30S sub-unit 8?12. The structure of Ksg bound to the E. coli 70S ribosome was determined by X-ray crystallography.The Ksg-binding pocket is composed entirely of 16S rRNA residues that line the mRNA channel of the small subunit, located between the universally conserved A794 and G926

Resistance to Ksg was first found to the ksgA gene that encodes a methyltransferase that catalyzes the post-transcriptional dimethylation of N6 of A1518 and A1519 in the loop that closes helix 45 near the 3' end of 16S rRNA.

II. General Structure

Ksg is an aminoglycoside antibiotic with the chemical formula C14H28CIN3O10.

CAP is a dimer of 22, 500 molecular weight, composed of two chemically identical polypeptide chains each 209 amino acids in length. The overall structure of the dimer is assymetric; one subunit adopts a "closed" conformation in which the amino- and carboxy-termini are closer together than in the more "open" subunit. Each subunit is composed of two distinct domains connected by a hinge region. The N-terminal domain is responsible for dimerization and cAMP binding. The carboxy-terminal domain contains a helix-turn helix DNA binding motif, and is also responsible for DNA bending. 

III. Ksg Binding Site

Ksg consists of an acetamidinium-carboxylate group attached to a hexopyrano-syl ring and an inositol ring. In the refined structure, the inositol ring packs against the bases of G926 and G1505, which are stacked. The O4- and O2-hydroxyls on the inositol ring are within hydrogen-bonding distance of the N1 of G926 and N7 of G1505, respectively.The hexopyranosyl ring of Ksg bridges the backbones of 16S rRNA nucleotides in the loop closed by bases A1499 and U1506. The hexopyranosyl methyl group may pack against the ribose C3' atom of G1505, whereas the amine makes direct contacts with the phosphate of A1499. The acetamidinium-carboxylate functional group of Ksg makes a number of contacts that are probably part of a water-mediated hydrogen bond network. In both ribosomes, the carboxy-imino N3 of Ksg is within hydrogen-bonding distance of the N1 of A794 and within van der Waals distance of the C2 of A794. The same atom of Ksg is also within van der Waals distance of the N6 of A792 in 30S-1 and possibly in 30S-2.

IV. DNA Binding

Once CAP has bound cAMP, it is ready to bind to the DNA. Binding occurs at the conserved sequence of 5'-AAATGTAGATCACATTT-3' Hydrogen bonds between the protein and the DNA phsophates occur at the backbone amide of residue 139, and the side chains of Thr-140, Ser-179, and Thr-182 In addition to these phosphate interactions, the side chains of Glu-181 and Arg-185, both emanating from the recognition helix directly contact the bases within the major groove of the DNA. Because of the way that the protein binds to the DNA, a kink of ~40 degrees occurs between nucleotide base pairs six and seven on each side of the dyad axis, 5'-TG-3' This sequence has been shown to favor DNA flexibility and bending in other systems as well. Because of this kink, an additional five ionic interactions and four hydrogen bonds are able to occur between the protein and the DNA strand. Examples of these new interactions occur between Lys-26, Lys-166, His-199 and the DNA sugar-phosphate backbone The DNA bend is integral to the mechanism of transcription activation. Not only does it place CAP in the proper orientation for interaction with RNA polymerase, but wrapping the DNA around the protein may result in direct contacts between upstream DNA and RNA polymerase. 

V. Activating Regions

Transcription activation by CAP requires more than merely the binding of cAMP and binding and bending of DNA. CAP contains an "activating region" that has been proposed to participate in direct protein-protein interactions with RNA polymerase and/or other basal transcription factors. Specifically, amino acids 156, 158, 159, and 162 have been proposed to be critical for transcription activation by CAP. These amino acids are part of a surface loop composed of residues 152-166 Researchers have concluded that the third and final step in transcription activation is this direct protein-protein contact between amino acids 156-162 of CAP, and RNA polymerase.

VI. References

Gunasekera, Angelo, Yon W. Ebright, and Richard H. Ebright. 1992. DNA Sequence Determinants for Binding of the Escherichia coli Catabolite Gene Activator Protein. The Journal of Biological Chemistry 267:14713-14720.

Schultz, Steve C., George C. Shields, and Thomas A. Steitz. 1991. Crystal Structure of a CAP-DNA complex: The DNA Is Bent by 90 degrees Science 253: 1001-1007.

Vaney, Marie Christine, Gary L. Gilliland, James G. Harman, Alan Peterkofsky, and Irene T. Weber. 1989. Crystal Structure of a cAMP-Independent Form of Catabolite Gene Activator Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Biochemistry 28:4568-4574.

Weber, Irene T., Gary L. Gilliland, James G. Harman, and Alan Peterkofsky. 1987. Crystal Structure of a Cyclic AMP-independent Mutant of Catabolite Activator Protein. The Journal of Biological Chemistry 262:5630-5636.

Zhou, Yuhong, Ziaoping Zhang, and Richard H. Ebright. 1993. Identification of the activating region of catabolite gene activator protein (CAP): Isolation and characterization of mutants of CAP specifically defective in transcription activation. Proceedings of the National Academy of Sciences of the United States of America 90:6081-6085.

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