TY - CHAP
T1 - Reconstitution of Mammalian 48S Ribosomal Translation Initiation Complex
AU - Majumdar, Romit
AU - Chaudhuri, Jayanta
AU - Maitra, Umadas
N1 - Funding Information:
Research in the author's laboratory was supported by Grant GM 15399 from the National Institutes of Health and by Cancer Core Support Grant P30CA13330 from the National Cancer Institute.
PY - 2007
Y1 - 2007
N2 - Initiation of translation is defined as the process by which a 40S ribosomal subunit, containing bound initiator methionyl-tRNA (Met-tRNAi), 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-tRNAi, 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-tRNAi 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.
AB - Initiation of translation is defined as the process by which a 40S ribosomal subunit, containing bound initiator methionyl-tRNA (Met-tRNAi), 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-tRNAi, 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-tRNAi 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.
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U2 - 10.1016/S0076-6879(07)30008-6
DO - 10.1016/S0076-6879(07)30008-6
M3 - Chapter
C2 - 17913639
AN - SCOPUS:38449112364
SN - 9780123739698
T3 - Methods in Enzymology
SP - 179
EP - 208
BT - Translation Initiation
PB - Academic Press Inc.
ER -