Gite S, RajBhandary U L
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 1997 Feb 21;272(8):5305-12. doi: 10.1074/jbc.272.8.5305.
The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in Escherichia coli. In attempts to identify regions of MTF that come close to the 3'-end of the tRNA, we oxidized 32P-3'-end-labeled E. coli initiator methionine tRNA with sodium metaperiodate and cross-linked it to MTF. The cross-linked MTF was separated from uncross-linked MTF by DEAE-cellulose chromatography, and the tRNA in the cross-linked MTF was hydrolyzed with nuclease P1 and RNase T1, leaving behind an oxidized fragment of [32P]AMP attached to MTF. Trypsin digestion of the cross-linked MTF followed by high pressure liquid chromatography of the digest yielded two peaks of radioactive peptides, I* and II*. These peptides were characterized by N- and/or C-terminal sequencing and by matrix-assisted laser desorption ionization mass spectroscopy. Peptide I* contained amino acids Gln186-Lys210 with Lys207 as the site of the cross-link. Peptide II*, a partial digestion product, contained amino acids Gln186-Arg214 also with Lys207 as the site of the cross-link. The molecular masses of peptides I* and II* indicate that the final product of the cross-linking reaction between the periodate-oxidized AMP moiety of the tRNA and Lys207 is most likely a morpholino derivative rather than a reduced Schiff's base.
甲硫氨酰 - tRNA甲酰基转移酶（MTF）对起始甲硫氨酰 - tRNA的特异性甲酰化作用对于大肠杆菌中蛋白质合成的起始至关重要。为了确定MTF中靠近tRNA 3'末端的区域，我们用偏高碘酸钠氧化了32P - 3'末端标记的大肠杆菌起始甲硫氨酸tRNA，并将其与MTF交联。通过DEAE - 纤维素色谱法将交联的MTF与未交联的MTF分离，交联的MTF中的tRNA用核酸酶P1和核糖核酸酶T1水解，留下附着在MTF上的[32P] AMP氧化片段。对交联的MTF进行胰蛋白酶消化，然后对消化产物进行高压液相色谱分析，得到两个放射性肽峰，I和II。通过N - 和/或C - 末端测序以及基质辅助激光解吸电离质谱对这些肽进行了表征。肽I包含氨基酸Gln186 - Lys210，交联位点为Lys207。肽II是一个部分消化产物，也包含氨基酸Gln186 - Arg214，交联位点同样为Lys207。肽I和II的分子量表明，tRNA的高碘酸盐氧化的AMP部分与Lys207之间交联反应的最终产物很可能是吗啉代衍生物，而不是还原的席夫碱。
