Kasugamycin Function
in Thermus thermophilus 30S
Ribosomal Subunit: Inhibition of Canonical Translation
Edna Kemboi '16 and Jiayu Chen '17
Contents:
I. Introduction
Kasugamycin
(Ksg) is an antibiotic produced by the
bacterium Streptomyces
kasugaensis. It inhibits proliferation of bacteria by
inhibiting initiation of translation by binding to 16s
rRNA, mimicking codon nucleotides at the
P-
site and E sites and
blocking initiator t-RNA
binding through perturbation of the mRNA-tRNA
codon-anticodon interaction during 30S canonical
initiation. The structure of Ksg bound
to the T. thermophilus
70S ribosome was determined by X-ray crystallography. This
tutorial mainly shows the structure of 30S
subunit of ribosome of T.
thermophilus. E.
coli numbering is used throughout the
whole tutorial.
II. Structure of Kasugamycin
Ksg is an aminoglycoside antibiotic with the chemical formula
C14H28CIN3O10.Click
Here for Structure of Ksg It consists of an acetamidiniumcarboxylate
group attached to an inositol
ring (ring I) and a hexopyranosyl
ring (ring II).
At both binding sites, the hexopyranosyl
ring bridges the backbone
of 16S
rRNAnucleotides.
The inositol ring forms hydrogen
bonds with oxygen and nitrogen atoms of
nucleotides. Furthermore, the acetaminiumcarboxylate
group makes a number of contacts that may involve in hydrogen bond network with
either nucleotide or amino acid of ribosomal proteins.
Click
Here for Schematics of Ksg1 and Ksg2 interactions with the
30S subunit.
III. Ksg Binding Site
The
Ksg-binding pocket is composed entirely of 16S
rRNA residues line the mRNA channel of
the 30S subunit and protect two nucleotides, the
universally conserved, A794
and G926 of
16S
rRNA. Also, kasugamycin
has two binding site: primary and
secondary binding sites.
Two
binding sites for Ksg are observed, both of which are on
the intersubunit side of the 30S subunit. In the primary
binding site, Ksg1
sits at the top of helix
44, spanning the region between h24
and h28
of the 16S
rRNAand interacts with G926 in h28
through ring I. Ring II rests on the backbone of a
single-stranded region (h44-45), connecting h44
and h45,
and the tail reaches towards
A792
and A794 in h24.
The D-inositol ring of Ksg1
forms hydrogen bonds with N1
and N6 of G926 in h28
and O2P of G1504 in the h44-45
linker, and ring II forms a hydrogen bond to O3
of G1505. The
kasugamine tail forms hydrogen bonds with N6
of A792
and N1 of A794
in h24
and is within hydrogen-bonding distance of the backbone of
A1499
in h44.
In the secondary position, Ksg2
interacts with the base of G693
in h23
by reaching across the mRNA
channel (structure does not show) and with the
backbone of Gly81-Gly82
in the beta-hairpin extension of the
ribosomal protein S7,
while the tail of Ksg2
forms hydrogen bonds with the O2
of U788
and O2P of U789
in h24.
IV. mRNA Binding and Inhibition of
Translation
Once Ksg binds to ribosome, its binding
sites occupy a position overlapping the kink in
the mRNA
between the P-site
and E-site codons.
Specifically, ring I of Ksg1
encroaches on the ribose in the +1
position of the P-site
codon, whereas the kasugamine
moiety (ring
II and tail)
reaches toward the phosphateoxygen and ribose in
the +2 and
+1
positions of the E-site
codon, respectively.
Ksg2
is positioned so that
ring II mimics the base of a nucleotide
at second (-2) position of the E-site
codon. (The results were based on
programming so there was no actual PDB.) Click
Here for binding of mRNA
(E-tRNA
and Ksg
do not show)
Ksg inhibit P-tRNA
binding in an indirect way. P-tRNA
protects A794
and G926
of rRNA from chemical modification and
the kink between the P-
and E-site
codons of the 16S 3' end mRNA
mimic is stabilized by a hydrogen bond
to the N1
of G926,
suggesting that the protection of this base by P-tRNA
indeed results indirectly from the
presence of mRNA. Though the position of
Ksg1
does not overlap with that of the P-tRNA,
it does overlap with both P- and E-codons.
The overlap disrupts the function of mRNA
channel and antiSD
sequence which stablize binding
between tRNA and mRNA, and makes tRNA
leave. Ksg2
overlaps E-codon,
competes with E-tRNA
and therefore reinforces the disrupt
effect on binding.
V. Ksg only Inhibit
Canonical Translation
Ksg only inhibits the
translation initiation of canonical
mRNAs but has no effect on that of leaderless
mRNA in
vivo. This is because the
overlap between the binding position
of Ksg and canonical
mRNAs is quite extensive in the P and
E sites,
whereas for initiation with leaderless
mRNA, which does not have an antiSD
sequence, the overlap is
reduced to only one nucleotide
position. Click
Here for comparison
VI. References
Frank
Schluenzen,
Chie Takemoto,
Daniel N
Wilson,
Tatsuya
Kaminishi,
Joerg M Harms,
Kyoko
Hanawa-Suetsugu,
Witold
Szaflarski,
Masahito
Kawazoe,
Mikako
Shirouzo, Knud
H Nierhaus,
Shigeyuki
Yokoyama &
Paola Fucini.
2006. The
antibiotic
kasugamycin
mimics mRNA
nucleotides to
destabilize
tRNA binding
and inhibit
canonical
translation
initiation. Nature
structural
&
molecular
biology Volume
13: 871-878.
Barbara
S Schuwirth, J Michael Day, Cathy W Hau, Gary R
Janssen, Albert E Dahlberg, Jamie H Doudna Cate, &
AntoŽn Vila-Sanjurjo. 2006. Structural analysis of
kasugamycin inhibition of translation. Nature
structural & molecular biology. Volume
13: 879-886
Daniel
N. Wilson. 2009. The A?Z of bacterial translation
inhibitors. Critical
Reviews in Biochemistry and Molecular Biology, 2009;44(6):
393-433.
Daniel
Boehringer, Heather C. O'Farrell, Jason P. Rife and
Nenad Ban. 2006. Structural Insights into
Methyltransferase KsgA Function in 30S Ribosomal
Subunit Biogenesis. .
J. Biol. Chem. 2012,
287:10453-10459.
Back to Top