Zhang Chun-Mei, Liu Cuiping, Slater Simon, Hou Ya-Ming
Department of Biochemistry and Molecular Biology, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA.
Nat Struct Mol Biol. 2008 May;15(5):507-14. doi: 10.1038/nsmb.1423. Epub 2008 Apr 20.
Cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii synthesizes Cys-tRNA(Cys) by an indirect pathway, whereby O-phosphoseryl-tRNA synthetase (SepRS) acylates tRNA(Cys) with phosphoserine (Sep), and Sep-tRNA-Cys-tRNA synthase (SepCysS) converts the tRNA-bound phosphoserine to cysteine. We show here that M. jannaschii SepRS differs from CysRS by recruiting the m1G37 modification as a determinant for aminoacylation, and in showing limited discrimination against mutations of conserved nucleotides. Kinetic and binding measurements show that both SepRS and SepCysS bind the reaction intermediate Sep-tRNA(Cys) tightly, and these two enzymes form a stable binary complex that promotes conversion of the intermediate to the product and sequesters the intermediate from binding to elongation factor EF-1alpha or infiltrating into the ribosome. These results highlight the importance of the protein binary complex for efficient synthesis of Cys-tRNA(Cys).
半胱氨酰 - tRNA(Cys)(Cys - tRNA(Cys))是翻译所必需的,通常由半胱氨酰 - tRNA合成酶(CysRS)合成。然而,詹氏甲烷球菌通过间接途径合成Cys - tRNA(Cys),即O - 磷酸丝氨酰 - tRNA合成酶(SepRS)用磷酸丝氨酸(Sep)使tRNA(Cys)酰化,而Sep - tRNA - Cys - tRNA合成酶(SepCysS)将与tRNA结合的磷酸丝氨酸转化为半胱氨酸。我们在此表明,詹氏甲烷球菌SepRS与CysRS不同,它招募m1G37修饰作为氨酰化的决定因素,并且对保守核苷酸的突变显示出有限的辨别能力。动力学和结合测量表明,SepRS和SepCysS都紧密结合反应中间体Sep - tRNA(Cys),并且这两种酶形成稳定的二元复合物,该复合物促进中间体转化为产物,并使中间体不与延伸因子EF - 1α结合或渗入核糖体。这些结果突出了蛋白质二元复合物对高效合成Cys - tRNA(Cys)的重要性。