The Human Elongation Factor 1 A-2 Gene (EEF1A2): Complete Sequence and Characterization of Gene Structure and Promoter Activity Claus Bischoff, 1,2 Søren Kahns, 1 Ann Lund, Helle F. Jørgensen, 3 Morten Præstegaard, 4 Brian F.C. Clark, 5 and Henrik Leffers * IMSB, University of Aarhus, Gustav Wieds vej 10 C, 8000 Århus C, Denmark; and * Afd. for Vækst og Reproduktion, Sektion GR-5064, Rigshospitalet, Blegdamsvej 9, 2100 København Ø, Denmark Received March 13, 2000; accepted May 22, 2000 The eukaryotic elongation factor 1 A (eEF1A, for- merly EF1) is a key factor in protein synthesis, where it promotes the transfer of aminoacylated tRNAs to the A site of the ribosome. Two differentially ex- pressed isoforms of eEF1A, designated eEF1A-1 and eEF1A-2, are found in mammals. Here we report the isolation and sequencing of the gene (HGMW-ap- proved symbol EEF1A2) coding for the human eEF1A-2 isoform. Furthermore, we characterize the gene struc- ture and the activity of the promoter. Isolation of over- lapping clones from human libraries revealed that the human eEF1A-2 gene spans approximately 10 kb and consists of eight exons. The intron– exon boundaries of human EEF1A2 and EEF1A1 are conserved, yet the gene of the eEF1A-2 isoform is larger than the eEF1A-1 gene because of enlarged introns. Primer extension analysis identified the predominant transcription start site 166 bp upstream of the AUG codon. The start site maps to an adenine located within a consensus initiator element. Sequencing of a 2-kb 5-flanking pro- moter region revealed no TATA element. However, several putative cis-regulatory elements were discov- ered. The 5-promoter activity was characterized by transient transfection experiments. Progressive dele- tions of the upstream promoter region defined a min- imal promoter region, ranging from 16 to 92, that is sufficient to drive transcription. © 2000 Academic Press INTRODUCTION The eukaryotic elongation factor 1 A, eEF1A (for- merly EF-1; Ad Hoc Nomenclature Subcommittee Re- port, 1996), plays a key role in eukaryotic protein syn- thesis (Merrick, 1992). In its active GTP-bound form, eEF1A mediates the transfer of aminoacylated tRNA to the A site of the ribosome. Upon cognate codon– anticodon recognition, GTP is hydrolyzed, and the in- active eEF1A  GDP complex is released. The guanine nucleotide exchange factor eEF1B mediates recycling of eEF1A by catalyzing the exchange of GDP with GTP. Several other functions have also been ascribed to eEF1A. It has been reported to interact with the cy- toskeleton (Barbarese et al., 1995), it binds to and bundles actin filaments (Yang et al., 1990), and it has a severing effect on microtubules (Shiina et al., 1994). Additionally, it has been associated with the mitotic spindle (Ohta et al., 1990). eEF1A has also been sug- gested to play a role in tRNA channeling in protein synthesis (Negrutskii et al., 1994), to be an activator of the phosphoinositol 4 kinase (Yang et al., 1993), to increase UV-induced transformation susceptibility (Tatsuka et al., 1992), and to be involved in the ubi- quitin-dependent degradation of N  -substituted pro- teins (Gonen et al., 1994). In conclusion, eEF1A seems to be a multifunctional protein coordinating several cellular functions. Characteristic of eEF1A expression is that multiple isoforms are expressed within a certain species. In mammals, two isoforms of eEF1A, termed eEF1A-1 and eEF1A-2, have been characterized. The two iso- forms are 92% identical at the amino acid level (Knud- sen et al., 1993), and they display equal activities in an in vitro translation assay (Kahns et al., 1998). How- ever, eEF1A-1 and eEF1A-2 are differentially ex- pressed. eEF1A-1 is expressed in all known tissues except for skeletal muscle, whereas eEF1A-2 expres- sion appears to be limited to skeletal muscle, heart, brain, and aorta (Kahns et al., 1998; Knudsen et al., Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF163763. 1 The first two authors contributed equally to this work. 2 Present address: Institute of Human Genetics, Vilhelm Meyers Alle ´ 240, 8000 Århus C, Denmark. 3 Present address: ICMB, University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JR, UK. 4 Present address: BioImage A/S, Mørkhøj Bygade 28, 2860 Søborg, Denmark. 5 To whom correspondence should be addressed. Telephone:+45 89425055. Fax: +45 86123178. E-mail: clark@biobase.dk. Genomics 68, 63–70 (2000) doi:10.1006/geno.2000.6271, available online at http://www.idealibrary.com on 63 0888-7543/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.