Kasugamycin
Function in E. coli 30S
Ribosomal Subunit: inhibition of translation
Edna Kemboi '16 and Jiayu Chen '17
Contents:
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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