cerevisiae) [19], and CARP2A (the gene coding for the acidic ribosomal protein, P2A, in Candida albicans) [20], were recently shown to use naturally occurring HTS assay non-AUG triplets as translation initiators. Moreover, the translational efficiency of non-AUG initiation is deeply affected (by up

to 32-fold) by nucleotides at the -3 to -1 relative positions, especially -3. AARuug (R denotes A or G; uug denotes a non-AUG initiation codon) appears to represent the most favorable sequence context [21]. A unique feature of the gene expression of ALA1 is that the mitochondrial form of AlaRS is initiated from two consecutive in-frame ACG codons, with the first being more robust [19, 22]. Redundant ACGs contain stronger initiation activities than does a single ACG [23]. This feature of recurrence of non-AUG initiator codons may in itself represent a novel mechanism to improve the overall efficiency of translation

[24]. To investigate if any other non-AUG triplets can act as initiator codons in yeast, a random triplet was introduced into ALA1 to replace the native initiation sites and screened. We show herein that except for AAG and AGG, all other non-AUG codons that differ from AUG by a single nucleotide can functionally substitute for the redundant ACG initiator codons of ALA1. These non-AUG initiator codons possessed different initiating activities MG-132 cost and exhibited different preferences for various sequence contexts. For example, GTG, a less-efficient non-AUG initiator codon in the context of ALA1, was one of the strongest non-AUG initiator codons in the context of GRS1. On the contrary,

ATA, a fairly active non-AUG initiator codon in the context of ALA1, was essentially inactive in the context of GRS1. Thus, every non-AUG initiator codon may have its own favorite sequence context in yeast. Methods Construction of various ALA1 and ALA1-lexA fusion constructs Cloning of the wild-type (WT) ALA1 gene in a low-copy-number yeast shuttle vector, pRS315, was previously Interleukin-2 receptor described [19]. A 5′-end truncated version of ALA1, extending from base pairs +54 to +2877 (relative to ATG1) was amplified by a polymerase chain reaction (PCR) and cloned in the XbaI/XhoI sites of pRS315, yielding pCW415. To mutate the repeating ACG initiator codons of ALA1, a short ALA1 sequence containing base pairs -250 to +54 was amplified by a PCR as an EagI-XbaI fragment and cloned into the appropriate sites of pBluescript II SK (+/-) (Stratagene, La Jolla, CA). Mutations were created by a PCR-based mutagenesis following the protocols provided by Stratagene. The repeating ACG triplets, ACG(-25)/ACG(-24), were first mutated to GGT(-25)/ACC(-24) to eliminate their initiating activities. A random triplet (designated here as “”NNN”") was then introduced to replace GGT(-25).