Elucidating mechanistic principles underpinning eukaryotic translation initiation using quantitative fluorescence methods.
Eukaryotic translation initiation is an intricate process involving at least 11 formally classified eIFs (eukaryotic initiation factors), which, together with the ribosome, comprise one of the largest molecular machines in the cell. Studying such huge macromolecular complexes presents many challenges which cannot readily be overcome by traditional molecular and structural methods. Increasingly, novel quantitative techniques are being used to further dissect such complex assembly pathways. One area of methodology involves the labelling of ribosomal subunits and/or eIFs with fluorophores and the use of techniques such as FRET (Förster resonance energy transfer) and FA (fluorescence anisotropy). The applicability of such techniques in such a complex system has been greatly enhanced by recent methodological developments. In the present mini-review, we introduce these quantitative fluorescence methods and discuss the impact they are beginning to have on the field.
Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princes Street, Manchester M1 7DN, UK.
Abigail L
AL
Stevenson
Pedro P
PP
Juanes
John E G
JE
McCarthy
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D039642:Eukaryotic Initiation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D010442:Peptide Chain Initiation, Translational; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054657:Ribosome Subunits / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D039642:Eukaryotic Initiation Factors; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D046912:Multiprotein Complexes; D012333:RNA, Messenger