Plant Science 154 2000 89 – 98
In vivo evaluation of the context sequence of the translation initiation codon in plants
Marcin Lukaszewicz
a,b,1
, Marc Feuermann
b,1
, Be´ne´dicte Je´rouville
b
, Arnaud Stas
b
, Marc Boutry
b,
a
Institute of Microbiology, Wroclaw Uni6ersity, Przybyszewskiego
63
,
51
-
148
Wroclaw, Poland
b
Unite´ de Biochimie Physiologique, Uni6ersite´ Catholique de Lou6ain, Place Croix du Sud
2
-
20
, B-
1348
Lou6ain-La-Neu6e, Belgium Received 8 October 1999; received in revised form 3 December 1999; accepted 3 January 2000
Abstract
Statistical analysis of the AUG initiation codon context in several plant organisms identified a nucleotide preference in some positions around the AUG. Sixteen AUG contexts were studied using transient expression in tobacco, maize and Norway spruce.
Besides the importance of A or G at position − 3, we revealed the role of positions − 2, − 1 for which AA or CC were found to be the best for tobacco and maize, respectively. GC positions + 4, + 5 were also found to be important in both tobacco and
maize. Finally, we identified a variation in context efficiency according to cell type, since A was better than G at position − 3 in tobacco leaf protoplasts, while both nucleotides were equally efficient in tobacco suspension cells. © 2000 Elsevier Science Ireland
Ltd. All rights reserved.
Keywords
:
Consensus AUG context; Dicot; Gymnosperm; Monocot; Plant; Translation context; Translation initiation www.elsevier.comlocateplantsci
1. Introduction
In eucaryotes, translation by cytosolic ribo- somes generally occurs at the first transcript AUG.
However, efficient recognition of an AUG codon as a translation initiation site depends on several
factors, such as the distance from the transcript 5 end, the secondary structure around the AUG
codon, and the nucleotide sequence flanking the translation initiation site [1 – 3]. The frequency of
nucleotides surrounding the initiation AUG con- text has been extensively analysed in genes avail-
able in databases [4], revealing a consensus sequence i.e. the most frequent nucleotides dis-
tinct for various groups of organisms. Thus Ya- mauchi [5] deduced the consensus sequence for
protozoa as U A A A AUG A N A U, with A at position − 3 being the best conserved nucleotide.
A at position − 3 was also shown to be a con- served nucleotide in Saccharomyces cere6isiae [6].
From the studies of 699 vertebrate AUG contexts, Kozak [7] deduced C AG C C AUG G to be a
consensus sequence. Finally, Lu¨tcke et al. [8], Joshi [9] and Cavener and Ray [10] made a similar
analysis for plant genes. Joshi [11] recently ex- tended this analysis to 5074 sequences allowing
identification of a consensus context for monocot ac AG AC c AUG G C and dicot plants a A
AC a AUG G C. In most cases, adenine at − 3 and guanine at + 4 position were found to be the
most frequent nucleotides.
The biological significance of the consensus se- quence derived from statistical analysis of data-
bases is less documented, especially in plants. Lu¨tcke et al. [8] tested the AUG context in both
the rabbit reticulocyte and the wheat germ transla- tion systems. Substituting A at position − 3 by G,
U or C significantly reduced expression in the
Corresponding author[f1]. Tel.: + 32-10-473621; fax: + 32-10- 473872.
E-mail address
:
boutryfysa.ucl.ac.be M. Boutry
1
These two authors contributed equally to this work. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 1 9 5 - 3
rabbit reticulocyte system, which was not the case in the wheat germ system, suggesting a possible
difference in the mechanism of translation initia- tion between plants and animals. Systematic muta-
genesis
experiments with
the vertebrate
preproinsulin gene expressed in transfected animal cells confirmed the importance of purine at posi-
tion − 3, as well as the importance of C at posi- tions − 2 and − 1 and G at position + 4 [12,13].
More recent in vitro and in vivo studies with influenza and parainfluenza viral RNA also sug-
gested the importance of positions + 4, + 5 and +
6 [14,15]. However, the role of the + 5 and + 6 positions was questioned recently by Kozak [16].
Experimental data also supported the importance of A at position − 3 in Saccharomyces cere6isiae
[17]. Transient expression in tobacco mesophyll protoplasts of the b-glucuronidase reporter with
two different AUG contexts: CCACC AUG G as an ‘optimal’ sequence for the rat preproinsuline
gene in COS cells and AACA AUG G as a plant consensus context did not show any significant
difference in translation efficiency between the two constructs [18]. Luehrsen and Walbot [19] studied
the effect of an upstream out-of-frame AUG codon that severely affected expression of the re-
porter gene, even though it was surrounded by a poor context, suggesting a lesser role for AUG
context in plants than in animals. On the other hand, the importance of AUG context in plants
was supported by a study showing a 4-fold im- provement of translation of a chitinase protein
when positions − 3 and + 4 were modified into A and G, respectively [20]. Similar results were ob-
served for two viral RNA [21,22] and the GUS reporter gene [23].
Much less is known about the impact of other positions and the interaction between them. Thus,
it is impossible to predict how efficiently a tran- script with an AUG initiation context different
from the consensus will be translated. Another interesting issue is how the consensus between
organisms diverged during evolution. For in- stance, does the distinct consensus determined for
the dicot and monocot plants reflect a modifica- tion in the translational machinery? In this study,
we have addressed these issues by comparing luci- ferase LUC expression produced from 16 gene
constructs with distinct AUG contexts introduced in Nicotiana tabacum, Zea mays and Picea abies
cells.
2. Methods
