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Volumn 283, Issue 5410, 1999, Pages 2097-2100

A cytotoxic ribonuclease targeting specific transfer RNA anticodons

Author keywords

[No Author keywords available]

Indexed keywords

ASPARAGINE TRANSFER RNA; ASPARTIC ACID TRANSFER RNA; COLICIN; HISTIDINE TRANSFER RNA; QUEUINE; RIBONUCLEASE; TRANSFER RNA; TYROSINE TRANSFER RNA;

EID: 0033605708     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5410.2097     Document Type: Article
Times cited : (148)

References (43)
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    • The plasmid producing E5-CRD and ImmE5 was constructed as follows. A Nco 1 site (CCATGG) was introduced into the initiation codon of the colE3 gene of pSH357 [H. Masaki, A. Akutsu, T. Uozumi, T. Ohta, Gene 107, 133 (1991)]. and the colE3-immE3 segment from this Nco 1 site to the Bcl 1 (TGATCA) site was replaced with a polymerase chain reaction (PCR)-amplified ColE5-099 DNA fragment, giving rise to plasmid pTO501, which produces the E5-CRD/ImmE5 complex in response to SOS inducers. For this purpose, an Nco 1 site was created at the 5′ end of the PCR fragment in such a way that the Asp at the 116th residue from the COOH-terminus of E5 was changed to an fMet, where the ATG is within the Nco 1 site. The termination codon of immE5 was changed to TGA to create a Bcl 1 site at the 3′ end of the PCR fragment.
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    • 2-terminal sequences of both ImmE5 proteins were identical and lacked fMet. Through similar construction, E3-CRD was produced from the truncated co/E3 gene encoding the COOH-terminal 110 amino acids plus an fMet, and the final product was confirmed to lack fMet.
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    • Figure 2B suggests almost the same size for the E3 fragment and the minor RNA band by E5-CRD, but in fact, just the 5′ end was not determined in the sequencing of the E5-CRD minor band (11). The new high-resolution gel suggests that the E5-CRD minor band is 1 base smaller (48 bases?) than the major E3 fragment (49 bases). The 3′ next sequence of the E3-sensitive site, A50-G49, is G49-U48 (numbers being from the 3′ end) in 16S rRNA. If E5-CRD preferentially cleaves this GU in 165 rRNA, this interestingly resembles the cleavage site, GU (QU), within the E5-sensitive tRNA anticodons (Fig. 3B).
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    • note
    • It was known that there is a great discrepancy between the RNA degradation and ribosorne inactivation activities of the E3-T2A fragment (almost the same as E3-CRD). Usually about 1 μg and 1 ng of E3-T2A were used for RNAs and ribosomes, respectively (12). Because the RNA degradation activity of ES-CRD was in fact higher than that of E3-CRD, the discrepancy of the concentrations used was reduced to about two orders of magnitude.
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    • note
    • We thank I. Nitta and M. Sano for technical advice; M. Hidaka, A. Nakamura, Y. Gunji, and J. Sagara for helpful discussions; and D. P. Brunner for ColE5-099. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture.


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