Majumdar Romit, Chaudhuri Jayanta, Maitra Umadas
Department of Cell Biology, Albert Einstein College of Medicine of Yeshiva University, Jack and Pearl Resnick Campus, Bronx, New York, USA.
Methods Enzymol. 2007;430:179-208. doi: 10.1016/S0076-6879(07)30008-6.
Initiation of translation is defined as the process by which a 40S ribosomal subunit, containing bound initiator methionyl-tRNA (Met-tRNA(i)), is positioned at the initiation AUG codon of an mRNA to form the 48S initiation complex. Subsequently, a 60S ribosomal subunit joins the 48S initiation complex to form an elongation-competent 80S initiation complex. By use of highly purified eukaryotic translation initiation factors (eIFs), ribosomes, Met-tRNA(i), mRNA, GTP as an effector molecule, and ATP as a source of energy, the initiation step of translation can be efficiently reconstituted. In this chapter, we describe the detailed procedure for efficient binding of Met-tRNA(i) to the 40S ribosomal subunit, the subsequent binding of the resulting 43S preinitiation complex to an mRNA, and scanning and positioning of the 43S complex at the AUG start codon of the mRNA to form the 48S initiation complex.
一个包含结合的起始甲硫氨酰 - tRNA（Met - tRNA(i)）的40S核糖体亚基定位在mRNA的起始AUG密码子处，形成48S起始复合物。随后，一个60S核糖体亚基加入48S起始复合物，形成具有延伸能力的80S起始复合物。通过使用高度纯化的真核翻译起始因子（eIFs）、核糖体、Met - tRNA(i)、mRNA、作为效应分子的GTP以及作为能量来源的ATP，翻译的起始步骤可以被有效地重建。在本章中，我们描述了Met - tRNA(i)与40S核糖体亚基高效结合的详细步骤，随后所得43S前起始复合物与mRNA的结合，以及43S复合物在mRNA的AUG起始密码子处的扫描和定位以形成48S起始复合物的过程。
