Scherf David, Hammermeister Alexander, Böhnert Pauline, Burkard Alicia, Helm Mark, Glatt Sebastian, Schaffrath Raffael
Institute of Biology, Division of Microbiology, University of Kassel, D-34132 Kassel, Germany.
Małopolska Centre of Biotechnology, Jagiellonian University, 30387 Krakow, Poland.
Nucleic Acids Res. 2025 Apr 10;53(7). doi: 10.1093/nar/gkaf296.
In yeast, tRNA modifications that are introduced by the Elongator complex are recognized by zymocin, a fungal tRNase killer toxin that cleaves the anticodon. Based on zymocin resistance conferred by mutations in KTI12, a gene coding for an Elongator interactor, we further examined the yet vaguely defined cellular role of Kti12. Guided by structural similarities between Kti12 and PSTK, a tRNA kinase involved in selenocysteine synthesis, we identified conserved basic residues in the C-terminus of Kti12, which upon site-directed mutagenesis caused progressive loss of tRNA binding in vitro. The inability of Kti12 to bind tRNA led to similar phenotypes caused by Elongator inactivation in vivo. Consistently, tRNA binding deficient kti12 mutants drastically suppressed Elongator dependent tRNA anticodon modifications and reduced the capacity of Kti12 to interact with Elongator. We further could distinguish Elongator unbound pools of Kti12 in a tRNA dependent manner from bound ones. In summary, the C-terminal domain of Kti12 is crucial for tRNA binding and Kti12 recruitment to Elongator, which are both requirements for Elongator function suggesting Kti12 is a tRNA carrier that interacts with Elongator for modification of the tRNA anticodon.
在酵母中，由延伸因子复合物引入的tRNA修饰可被zymocin识别，zymocin是一种真菌tRNase杀伤毒素，可切割反密码子。基于KTI12（一种编码延伸因子相互作用蛋白的基因）突变赋予的zymocin抗性，我们进一步研究了Kti12尚未明确的细胞功能。根据Kti12与参与硒代半胱氨酸合成的tRNA激酶PSTK之间的结构相似性，我们在Kti12的C末端鉴定出保守的碱性残基，定点诱变后这些残基导致体外tRNA结合能力逐渐丧失。Kti12无法结合tRNA导致体内延伸因子失活引起的类似表型。一致地，缺乏tRNA结合能力的kti12突变体极大地抑制了延伸因子依赖性tRNA反密码子修饰，并降低了Kti12与延伸因子相互作用的能力。我们还能以tRNA依赖的方式区分未与延伸因子结合的Kti12池和已结合的Kti12池。总之，Kti12的C末端结构域对于tRNA结合以及Kti12募集到延伸因子至关重要，这两者都是延伸因子功能的必要条件，表明Kti12是一种tRNA载体，与延伸因子相互作用以修饰tRNA反密码子。
