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. 1991 May;1(2):111-5.

A short leader sequence impairs the fidelity of initiation by eukaryotic ribosomes

M Kozak  1
Affiliations

A short leader sequence impairs the fidelity of initiation by eukaryotic ribosomes

M Kozak. Gene Expr. 1991 May.

Abstract

The functional consequences of unusually short 5' noncoding sequences on eukaryotic mRNAs are explored here by using an in vitro transcription and translation system. As the distance of the first AUG codon from the m7G cap was decreased from 32 to 3 nucleotides, the yield of protein initiated from the first AUG codon progressively decreased, with a corresponding increase in initiation from the second AUG codon. The leakiness attributable to a too-short leader sequence was offset, however, by introducing secondary structure downstream from the first AUG codon.

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Figures

Figure 1
Figure 1
T7-generated transcripts with short leader sequences. The four plasmids in this series are designated T3, T6, T9, and T12, where the numbers indicate the lengths of the leader sequences when the plasmids are transcribed by T7 RNA polymerase. The oligonucleotide insert that carries the bacteriophage T7 promoter is shown in lower case letters in the top line. Below, the beginning of the transcribed sequence is shown, with T’s in place of U’s, for all four constructs. Initiation at the SP6 promoter, which was retained upstream from the T7 promoter, produces mRNAs with leader sequences 29 nucleotides longer than those on the corresponding T7 transcripts; the SP6-generated mRNAs from this series were used only where explicitly stated in the text. In addition to the authentic AUG initiator codon for CAT protein, each transcript has an upstream AUG triplet that enables synthesis of an N-terminally extended “preCAT” protein. To adjust the reading frame between the two AUG codons, a 22-nucleotide adaptor (upper line, bracketed) was inserted. Adaptor 8336 introduces a stem-loop structure (AG -19 kcal/mol) downstream from the preCAT start site; the alternative adaptor 8334 has no deliberate secondary structure.
Figure 2
Figure 2
Demonstration that short leader sequences encourage leaky scanning in the wheat germ translation system. [:«S]methionine-labeled proteins were fractionated by polyacrylamide gel electrophoresis as described previously (Kozak, 1989). The yield of CAT protein (lower band in the autoradiogram) is a measure of the extent to which ribosomes bypass the first AUG codon and initiate instead at the second AUG. With T7-derived mRNAs from plasmids T3(8334), T6(8334), T9(8334), and T12(8334), initiation from the second AUG codon declined as the 5′ non-coding sequence was lengthened from 3 to 12 nucleotides (lanes 1–4). In lanes 6–9, the presence of the structure-prone oligonucleotide 8336 downstream from the preCAT start site suppressed the tendency of ribosomes to bypass the first AUG codon.
Figure 3
Figure 3
Initiation of translation from the first and second AUG codons as a function of leader length. T7-derived transcripts from plasmids T3(8334), T6(8334), T9(8334), and T12(8334) were translated in the wheat germ system. T3(8334) was also transcribed with SP6 polymerase to obtain the indicated 32-nucleotide leader sequence. [35S]methionine-labeled preCAT (•) and CAT (○) proteins were fractionated by polyacrylamide gel electrophoresis as in Figure 2; the resulting autoradiogram was quantified by densitometry. Protein yields in optical density units are plotted as a function of distance from the m7G cap to the preCAT start site (AUG #1).
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