In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.
Translation initiation starts with the formation of the 43S preinitiation complex (PIC) consisting of several soluble factors, including the ternary complex (TC; elF2-GTP-Met-tRNA(i)(Met)), which associate with the small ribosomal subunit. In the next step, mRNA is recruited to form the 48S PIC and the entire machinery starts scanning the 5' untranslated region of the mRNA until the AUG start codon is encountered. The most widely used method to separate 40S and 60S ribosomal subunits from soluble factors, monosomes and polysomes, is sucrose density centrifugation (SDC). Since PICs are intrinsically unstable complexes that cannot withstand the forces imposed by SDC, a stabilization agent must be employed to detect the association of factors with the 40S subunit after SDC. This was initially achieved by adding heparin (a highly sulfated glycosaminoglycan) directly to the breaking buffer of cells treated with cycloheximide (a translation elongation inhibitor). However, the mechanism of stabilization is not understood and, moreover, there are indications that the use of heparin may lead to artifactual factor associations that do not reflect the factor occupancy of the 43S/48S PICs in the cell at the time of lysis. Therefore, we developed an alternative method for PIC stabilization using formaldehyde (HCHO) to cross-link factors associated with 40S ribosomal subunits in vivo before the disruption of the yeast cells. Results obtained using HCHO stabilization strongly indicate that the factors detected on the 43S/48S PIC after SDC approximate a real-time in vivo "snapshot" of the 43S/48S PIC composition. In this chapter, we will present the protocol for HCHO cross-linking in detail and demonstrate the difference between heparin and HCHO stabilization procedures. In addition, different conditions for displaying the polysome profile or PIC analysis by SDC, used to address different questions, will be outlined.
Valásek
Leos
L
Institute of Microbiology, AS CR, Prague, Czech Republic.
Szamecz
Bela
B
Hinnebusch
Alan G
AG
Nielsen
Klaus H
KH
D015152:Blotting, Northern; D015153:Blotting, Western; D002457:Cell Extracts; D002458:Cell Fractionation / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D039642:Eukaryotic Initiation Factors / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry; D010442:Peptide Chain Initiation, Translational / Q000502:physiology*; D011132:Polyribosomes / Q000502:physiology
D002457:Cell Extracts; D003432:Cross-Linking Reagents; D039642:Eukaryotic Initiation Factors; D005557:Formaldehyde; D006493:Heparin