Protection-based assays to measure aminoacyl-tRNA binding to translation initiation factors.
To decipher the mechanisms of translation initiation, the stability of the complexes between tRNA and initiation factors has to be evaluated in a routine manner. A convenient method to measure the parameters of binding of an aminoacyl-tRNA to an initiation factor results from the property that, when specifically complexed to a protein, the aminoacyl-tRNA often resists spontaneous deacylation. This chapter describes the preparation of suitable aminoacyl-tRNA ligands and their use in evaluating the stability of their complexes with various initiation factors, such as e/aIF2 and e/aIF5B. The advantages and the limitations of the method are discussed.
Mechulam
Yves
Y
Laboratoire de Biochimie, CNRS Ecole Polytechnique, Palaiseau Cedex, France.
Guillon
Laurent
L
Yatime
Laure
L
Blanquet
Sylvain
S
Schmitt
Emmanuelle
E
D039642:Eukaryotic Initiation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000302:isolation & purification / Q000378:metabolism
D039642:Eukaryotic Initiation Factors; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met