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Blood, 1 February 2003, Vol. 101, No. 3, pp. 1202-1202
CORRESPONDENCE
To the editor:
Not every polymorphism close to the AUG codon can be explained
by invoking context effects on initiation of translation
The  1C>T change in the annexin V gene, which reduces
the risk of myocardial infarction,1 cannot be explained as
simply as your editorial note suggests.2 The demonstration by Gonzalez-Conejero et al1 that
1T increases the efficiency of translation might be correct (the
fourth paragraph of this letter discusses some concerns about the in vitro translation assay), but if so, the effect would not be explicable by invoking the Kozak context rules. The rules predict the opposite of
what was seen; that is, translation should be more efficient with C
rather than T in position 1, if that position scores at all. GCCRCCaugG (R = purine) is the optimal context for recognition of the
start codon in mammals. Within this motif, some positions are more
important than others. Mutagenesis experiments with laboratory constructs showed that A or G in position 3 (3 nt upstream from the
AUG codon, which is numbered +1 to +3) and G in position +4 make the
strongest contributions.3,4 Only in the absence of 3R
and +4G do mutations in positions 1 and 2 score
strongly.3 Inasmuch as the annexin V start site conforms
to the consensus motif in positions 3 and +4, the identity of the
base in position 1 would be expected to affect translation only
slightly, if at all. The context rules, initially established by studying translation in
cultured cells,3,5 can be replicated using in vitro translation systems, but the experiments must be designed carefully. Reaction conditions, such as the concentration of
magnesium,4,6 can profoundly affect whether recognition of
the AUG codon in vitro displays the same sensitivity to context as is
seen in vivo. The coupled transcription/translation system used by many
investigators, including Gonzalez-Conejero et al,1 makes
it difficult to adjust magnesium levels and to be sure that exactly the
same amount of mRNA is generated from each allele. A small change in
translational efficiency is believable when the reduction in initiation
from the first AUG codon is accompanied by an increase in initiation from the next AUG downstream (eg, Figure 1 in Kozak7) or
when an assay is used that directly monitors the initiation
step.4 One cannot be as confident, however, about a small
(1.4-fold) difference in translational efficiency based on measurement
of proteins precipitable by trichloroacetic acid.1 In addition to these practical complications in testing for context
effects, there are theoretical limitations. Because secondary structure
downstream from the AUG codon can compensate for a less than perfect
context,8 not every mutation within the consensus motif
will score. In some human and mouse genes, a mutation or polymorphism close
to the AUG codon, usually in position 3 or +4, has been shown to
reduce translational efficiency, with pathological
consequences.9 Only one previous example involves a change
in position 1: in a patient with ataxia with vitamin E deficiency, a
C>T mutation in the alpha-tocopherol transfer protein gene causes a
2-fold decrease in translation, measured in vivo.10 In
addition to these pathologies linked to mutations in the consensus
motif, there is a growing list of human diseases wherein translation of
a critical regulatory gene is perturbed by restructuring the 5' UTR in
ways that add or remove upstream AUG codons.9,11 Thus, the
scanning mechanism for initiation of translation does provide a
framework for understanding how some mutations cause disease. It is possible that the demonstrated increase in plasma levels of
annexin V protein associated with the 1T allele1
reflects an effect on mRNA stability or splicing. If followup studies
rule out these alternative explanations and confirm that the 1T
allele indeed augments translation, the reason could conceivably
involve an effect on mRNA secondary structure. I do not think this
particular polymorphism can be explained by invoking conventional
context effects on AUG codon recognition.
Marilyn Kozak
Correspondence: Department of Biochemistry, Robert
Wood Johnson Medical School, 675 Hoes Ln, Piscataway, NJ 08854; e-mail:
kozakma{at}umdnj.edu
References
1.
Gonzalez-Conejero R, Corral J, Roldan V, et al.
A common polymorphism in the annexin V Kozak sequence (1C>T) increases translation efficiency and plasma levels of annexin V, and decreases the risk of myocardial infarction in young patients.
Blood.
2002;100:2081-2086[Abstract/Free Full Text].
2.
Neufeld E.
Starting out right: Kozak sequences and clots.
Blood.
2002;100:1933[Free Full Text].
3.
Kozak M.
Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.
Cell.
1986;44:283-292[CrossRef][Medline]
[Order article via Infotrieve].
4.
Kozak M.
Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6.
EMBO J.
1997;16:2482-2492[CrossRef][Medline]
[Order article via Infotrieve].
5.
Kozak M.
At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells.
J Mol Biol.
1987;196:947-950[CrossRef][Medline]
[Order article via Infotrieve].
6.
Kozak M.
Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources.
Nucleic Acids Res.
1990;18:2828[Free Full Text].
7.
Kozak M.
Initiation of translation in prokaryotes and eukaryotes.
Gene.
1999;234:187-208[CrossRef][Medline]
[Order article via Infotrieve].
8.
Kozak M.
Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes.
Proc Natl Acad Sci U S A.
1990;87:8301-8305[Abstract/Free Full Text].
9.
Kozak M.
Emerging links between initiation of translation and human diseases.
Mammalian Genome.
2002;13:401-410[CrossRef][Medline]
[Order article via Infotrieve].
10.
Usuki F, Maruyama K.
Ataxia caused by mutations in the alpha-tocopherol transfer protein gene.
J Neurol Neurosurg Psychiatry.
2000;69:254-256[Abstract/Free Full Text].
11.
Kozak M.
Pushing the limits of the scanning mechanism for initiation of translation.
Gene.
2002;299:1-34[CrossRef][Medline]
[Order article via Infotrieve].
Response:
Intricacies of initiator polymorphism studies
I appreciate Dr Kozak setting the record straight on
technical pitfalls and interpretation of studies on initiation of
transcription related to polymorphisms close to the initiator
methionine. Her detailed critique should be borne in mind while
considering the annexin V polymorphism study of Gonzalez-Conejero et al
and related studies.
Ellis Neufeld
Correspondence: Children's
Hospital, Hematology Division, 300 Longwood Ave, Enders 7, Boston, MA
02115; e-mail:
ellis.neufeld{at}tch.harvard.edu
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