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.

Institute of Microbiology AS CR Prague Czech Republic

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Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes

In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation

Translation initiation factor eIF3 promotes programmed stop codon readthrough

Functional and biochemical characterization of human eukaryotic translation initiation factor 3 in living cells

Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes

Reporters transiently transfected into mammalian cells are highly sensitive to translational repression induced by dsRNA expression

Translation initiation factors eIF3 and HCR1 control translation termination and stop codon read-through in yeast cells

Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit

Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly

The eIF3c/NIP1 PCI domain interacts with RNA and RACK1/ASC1 and promotes assembly of translation preinitiation complexes

The RNA recognition motif of eukaryotic translation initiation factor 3g (eIF3g) is required for resumption of scanning of posttermination ribosomes for reinitiation on GCN4 and together with eIF3i stimulates linear scanning

eIF3a cooperates with sequences 5' of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA