Protein synthesis in eukaryotes normally begins at the first AUG codon in the mRNA. Sometimes, however, the ribosomes do not begin protein synthesis at this first AUG but scan past it (leaky scanning), and protein synthesis begins instead at an internal AUG. In order to understand what features of an mRNA affect the efficiency of initiation at the first AUG, studies have been undertaken in which the synthesis of chloramphenicol acetyltransferase was examined. Translation of its message can give rise to a protein referred to as preCAT or a slightly smaller protein, CAT (see M. Kozak, 2005, Gene 361:13). The two proteins differ in that CAT lacks several amino acids found at the N-terminus of preCAT. CAT is derived not by cleavage of preCAT, but instead by initiation of translation of the mRNA at an internal AUG:
a. Results from a number of studies have given rise to the hypothesis that the sequence (−3)ACCAUGG(+4), in which the start codon AUG is shown in boldface, provides an optimal context for initiation of protein synthesis and ensures that ribosomes do not scan past this first AUG to begin initiation instead at a downstream AUG. In the numbering scheme used here, the A of the AUG start codon is designated (+1); bases 5′ of this are given negative numbers [so that the first base of this sequence is (−3)], and bases 3′ to the (+1) A are given positive numbers [so that the last base of this sequence is (+4)]. To test the hypothesis that the start site sequence (−3)ACCAUGG(+4) prevents leaky scanning, the chloramphenicol acetyltransferase mRNA sequence was modified and the resulting effects on translation assessed. In the following figure, the sequence (red) surrounding the first AUG codon (black) of the mRNA that gives rise to the synthesis of preCAT is shown above lane 3. Modification of this message is shown above the other gel lanes (altered nucleotides are in blue), and the completed proteins generated from each modified message appear as bands on the SDS-polyacrylamide gel below. The intensity of each band is an indication of the amount of that protein synthesized. Analyze the alterations to the wild-type sequence and describe how they affect translation. Are the positions of some nucleotides more important than others? Do the data shown in this figure provide support for the hypothesis that the context in which the first AUG is present affects efficiency of translation from this site? Is ACCAUGG an optimal context for initiation from the first AUG?
The data suggest that context matters and that a change in the sequence surrounding the first AUG affects the efficiency of initiation from this start site. When comparing lanes 1 and 2, in which the mRNAs differ only at position (+4), one observes that a G, rather than a U, at this position reduces leaky scanning. More preCAT and less CAT is synthesized with the message used in lane 2 than with that used in lane 1. Although (−3)ACCAUGG(+4) is hypothesized to provide an optimal context in which the first AUG is presented, the data suggest that this sequence can be modified to ACCAUGA without significantly compromising efficiency of initiation from the first AUG. The only difference between the messages in lanes 3 and 4 is a change from G to A at position (+4), and in each case only preCAT is synthesized. With respect to the importance of ACC at positions (−3) to (−1), a comparison of lanes 4 and 5 reveal that a shift of ACC from position (−3) to (−1) (lane 4) to position (−4) to (−2) (lane 5) results in a loss of fidelity of initiation from the first start site. Replacement of (−3)ACC(−1) with (−3)UUU(−1) (compare lane 2 to lane 3) results in a loss of efficiency of translation from the first start site, so these data suggest that the ACC sequence and its position relative to the AUG matter.
b. What are some additional alterations to this message, other than those shown in the figure, that would further elucidate the importance of the ACCAUGG sequence as an optimal context for synthesis of preCAT rather than CAT? How would you further examine whether A at the (−3) position and G at the (+4) position are the most important nucleotides to provide context for the AUG start?
_feedback: In order to further test the importance of the nucleotide at the +4 position, it would be useful to undertake an analysis of CAT mRNA mutants with only single substitutions compared to the wild-type sequence shown in lane 3. The data shown provide evidence that G can be substituted with A (lane 4) at position (+4) and suggest that it cannot be substituted with U (lane 1). However, there are additional sequence changes in the mRNA in lane 1 other than the change at position (+4). It would be informative to change the mRNA used in lane 3 so that either U or C is substituted for G at position (+4). If each of these substitutions results in synthesis of some CAT, then one could deduce that an optimal context for the first start site can tolerate purines but not pyrimidines at the (+4) position. The data shown in the figure also do not examine the importance of the (−3)ACC(−1) sequence other than to change it completely to UUU. It would also be useful to change this sequence one nucleotide at a time to determine the relative importance of each of these nucleotides in helping the ribosomes pause at the first AUG site and begin translation. In each case, synthesis of CAT would be evidence that the particular substitution to the sequence results in a loss of fidelity of initiation at the first AUG. If the A at position (−3) is the most important of the ACC sequence for generating efficiency of translation for the first AUG, then one would expect that changes to this nucleotide would result in synthesis of more CAT than would changes to the other two nucleotides.
c. A mutation causing a severe blood disease has been found in a single family (see T. Matthes et al., 2004, Blood 104:2181). The mutation, shown in red in the figure below, has been mapped to the 5′-untranslated region of the gene encoding hepcidin and has been found to alter the gene’s mRNA. The shaded regions indicate the coding sequence of the normal and mutant genes. No hepcidin is produced from the altered mRNA, and lack of hepcidin results in the disease. Can you provide a reasonable explanation for the lack of synthesis of hepcidin in the family members who have inherited this mutation? What can you deduce about the importance of the context in which the start site for initiation of protein synthesis occurs in this case?
_feedback: The mutation in this family results in the introduction of a new AUG sequence in the hepcidin mRNA upstream from the original start site. Because the new AUG is not in frame with respect to the original start AUG, no hepcidin will be made if initiation begins exclusively at this new, upstream AUG. The fact that hepcidin is not synthesized in individuals who have inherited this mutation suggests that initiation of protein synthesis occurs at the new AUG with high efficiency and that the ribosomes do not scan through this site to begin synthesis downstream at the original start site. Thus, these findings support the hypothesis that initiation of protein synthesis in eukaryotes normally begins at the first 5’ AUG site. An examination of the context in which this new AUG start site is located reveals that it has the important G located at (+4), as does the original AUG start site. Whereas the original AUG has an A at the (−3) position (and, in fact, also has the consensus C at position (−2)), the new, upstream AUG does not have the consensus sequence in this position. However, given that no hepcidin is made, the new start site may be in a context that facilitates efficient recognition by the ribosomes and thereby does not result in any (or in any detectable) leaky scanning.
