Use of reticulocyte lysates for mechanistic studies of eukaryotic translation initiation.
This chapter describes how commercially available, nuclease-treated rabbit reticulocyte lysates can be used to study different types of translation initiation (cap-dependent initiation, reinitiation, internal ribosome entry site-mediated initiation) and the influence of different initiation factors on these translation mechanisms. Additionally, with the use of sucrose gradients, it is possible to use nuclease-treated reticulocyte lysates to monitor the formation of ribosomal complexes for their content of mRNA, initiator met-tRNA(i), and initiation factors. The advantage of using nuclease-treated lysates rather than purified initiation factors is that reactions occur at or near the in vivo rate in contrast to rates observed in reactions with purified components, which are generally 10- to 1000-fold lower. The disadvantage is not being able to accurately control the amount of individual initiation factors, although the use of either factor additions or specific inhibitors can be helpful in assessing the role of specific individual initiation factors.
Merrick
William C
WC
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
Barth-Baus
Diane
D
D000818:Animals; D002457:Cell Extracts; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D008836:Micrococcal Nuclease / Q000502:physiology; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification; D012343:RNA, Transfer / Q000302:isolation & purification; D011817:Rabbits; D011933:Reagent Kits, Diagnostic; D012156:Reticulocytes / Q000187:drug effects; D012270:Ribosomes / Q000378:metabolism
D002457:Cell Extracts; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic; D012343:RNA, Transfer; D008836:Micrococcal Nuclease