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Molecular and Cellular Biology, December 2003, p. 8925-8933, Vol. 23, No. 24
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.24.8925-8933.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Assembly of 48S Translation Initiation Complexes from Purified Components with mRNAs That Have Some Base Pairing within Their 5' Untranslated Regions

Sergei E. Dmitriev,1 Ilya M. Terenin,1 Yan E. Dunaevsky,1 William C. Merrick,2 and Ivan N. Shatsky1*

A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia,1 Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-49352

Received 23 May 2003/ Returned for modification 30 June 2003/ Accepted 11 August 2003

The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, it has been successful mostly for formation of 48S translation initiation complexes with viral IRES elements. Among cap-dependent mRNAs, only globin mRNAs and transcripts with artificial 5' leaders were amenable to this assembly. Here, with modified conditions for the reconstitution, 48S complexes have been successfully assembled with the 5' UTR of beta-actin mRNA (84 nucleotides) and the tripartite leader of adenovirus RNAs (232 nucleotides), though the latter has been able to use only the scanning rather then the shunting model of translation initiation with canonical initiation factors. We show that initiation factor 4B is essential for mRNAs that have even a rather moderate base pairing within their 5' UTRs (with the cumulative stability of the secondary structure within the entire 5' UTR < -13 kcal/mol) and not essential for beta-globin mRNA. A recombinant eIF4B poorly substitutes for the native factor. The 5' UTRs with base-paired G residues reveal a very sharp dependence on the eIF4B concentration to form the 48S complex. The data suggest that even small variations in concentration or activity of eIF4B in mammalian cells may differentially affect the translation of different classes of cap-dependent cellular mRNAs.

* Corresponding author. Mailing address: Belozersky Institute of Physico-Chemical Biology, Moscow State University, Bldg. "A", Moscow 119899, Russia. Phone: 095 9394857. Fax: 095 9393181. E-mail: shatsky{at}libro.genebee.msu.su.

Molecular and Cellular Biology, December 2003, p. 8925-8933, Vol. 23, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.24.8925-8933.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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