TY - JOUR
T1 - Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2
AU - Kitahara, Kei
AU - Kajiura, Akimasa
AU - Sato, Neuza Satomi
AU - Suzuki, Tsutomu
N1 - Funding Information:
We are grateful to the Suzuki lab members for many fruitful discussions and technical advices. We would also like to thank Dr Catherine L. Squires for providing us with valuable strains and plasmids. This work was supported by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of Japan (to T.S.) and the Human Frontier Science Program (RGY23/2003) (to T.S.). Funding to pay the Open Access publication charges for this article was provided by the Japan Ministry of Education, Science, Sports and Culture.
PY - 2007/6
Y1 - 2007/6
N2 - Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.
AB - Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.
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U2 - 10.1093/nar/gkm356
DO - 10.1093/nar/gkm356
M3 - Article
C2 - 17553838
AN - SCOPUS:34547619912
VL - 35
SP - 4018
EP - 4029
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 12
ER -