Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region.

A unique genetic selection was devised at the HIS4 locus to address the mechanism of translation initiation in Saccharomyces cerevisiae and to probe sequence requirements at the normal translational initiator region that might participate in ribosomal recognition of the AUG start codon. The first AUG codon at the 5' end of the HIS4 message serves as the start site for translation, and the -3 and +4 nucleotide positions flanking this AUG (AXXAUGG) correspond to a eucaryotic consensus start region. Despite this similarity, direct selection for mutations that reduce or abolish ribosomal recognition of this region does not provide any insight into the functional nature of flanking nucleotides. The only mutations identified that affected recognition of this region were alterations in the AUG start codon. Among 150 spontaneous isolates, 26 were shown to contain mutations in the AUG start codon, including all +1 changes (CUG, GUG, and UUG), all +3 changes (AUA, AUC, and AUU), and one +2 change (ACG). These seven mutations of the AUG start codon, as well as AAG and AGG constructed in vitro, were assayed for their ability to support HIS4 expression. No codon other than AUG is physiologically relevant to translation initiation at HIS4 as determined by growth tests and quantitated in his4-lacZ fusion strains. These data and analysis of other his4 alleles are consistent with a mechanism of initiation at HIS4 as proposed in the scanning model whereby the first AUG codon nearest the 5' end of the message serves as the start site for translation and points to the AUG codon in S. cerevisiae as an important component for ribosomal recognition of the initiator region.     

Sequence and structural features associated with translational initiator regions in yeast--a review

We have compared the translational initiator regions of 131 yeast genes. 95% utilize the first AUG from the 5' end of the message as the start codon for translation. Yeast leader regions in general are rich in adenine nucleotides (nt), have an average length of 52 nt, and are void of significant secondary structure. Sequences immediately adjacent to AUG start codons are preferred, however, the bias in nucleotide distribution (5'-A-YAA-UAAUGUCU-3') does not reflect a higher eukaryotic consensus (5'-CACCAUGG-3') with the exception of an adenine nucleotide preference at the -3 position. A minority of yeast mRNAs that contain AUG codons in the leader region that do not serve as the start codon for the primary gene product differ from the majority of mRNAs by one or more of these general properties. This analysis appears to indicate that basic features associated with yeast leader regions are consistent with a general mechanism of initiation of protein synthesis in eukaryotes, as proposed by the ribosomal 'scanning' model, but perhaps only basic features associated with ribosomal recognition of an AUG start codon are intact.     

AUG is the only initiation codon in eukaryotes

An analysis of mutants of the yeast Saccharomyces cerevisiae indicates that AUG is the sole codon capable of initiating translation of iso-1-cytochrome c. This result with yeast and the sequence results of numerous eukaryotic genes indicate that AUG is the only initiation codon in eukaryotes; in contrast, results with Escherichia coli and bacteriophages indicate that both AUG and GUG are initiation codons in prokaryotes. The difference can be explained by the lack of the t6 A hypermodified nucleoside (N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]threonine) in prokaryotic initiator tRNA and its presence in eukaryotic initiator tRNA.     

Mutational analysis of the HIS4 translational initiator region in Saccharomyces cerevisiae.

We have mutated various features of the 5' noncoding region of the HIS4 mRNA in light of established Saccharomyces cerevisiae and mammalian consensus translational initiator regions. Our analysis indicates that insertion mutations that introduce G + C-rich sequences in the leader, particularly those that result in stable stem-loop structures in the 5' noncoding region of the HIS4 message, severely affect translation initiation. Mutations that alter the length of the HIS4 leader from 115 to 39 nucleotides had no effect on expression, and sequence context changes both 5' and 3' to the HIS4 AUG start codon resulted in no more than a twofold decrease of expression. Changing the normal context at HIS4 5'-AAUAAUGG-3' to the optimal sequence context proposed for mammalian initiator regions 5'-CACCAUGG-3' did not result in stimulation of HIS4 expression. These studies, in conjunction with comparative and genetic studies in S. cerevisiae, support a general mechanism of initiation of protein synthesis as proposed by the ribosomal scanning model.     

The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream codons.

The selection of the site for initiation of translation for the Saccharomyces cerevisiae NFS1 gene was examined using mutated AUG1, AUG2 and AUG3 codons. When AUG1 of the yeast NFS1 gene was mutated to UUG and the resulting mRNA was translated in vitro using a reticulocyte system, initiation from the mutated codon was abolished and occurred instead at downstream codons at increased rates. When the same mRNA was translated using a yeast extract, translation initiated at the mutated codon, albeit at a reduced rate, and there was no increased translation at downstream AUG codons. The NFS1 gene in which AUG1 was replaced by UUG was also able to substitute for the wild-type gene in vivo in yeast. Western blots confirmed that the encoded protein was the same size as that encoded by the wild-type gene and that both the wild-type and mutated proteins localized to mitochondria. This is apparently the first example of a yeast protein where mutagenesis of AUG1 does not lead to alternate use of a downstream AUG.     

Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational machinery

Translation initiation in eukaryotes involves more than a dozen protein factors. Alterations in six factors have been found to reduce the fidelity of start codon recognition by the ribosomal preinitiation complex in yeast, a phenotype referred to as Sui(-). No small molecules are known that affect the fidelity of start codon recognition. Such compounds would be useful tools for probing the molecular mechanics of translation initiation and its regulation. To find compounds with this effect, we set up a high-throughput screen using a dual luciferase assay in S. cerevisiae. Screening of over 55,000 compounds revealed two structurally related molecules that decrease the fidelity of start codon selection by approximately twofold in the dual luciferase assay. This effect was confirmed using additional in vivo assays that monitor translation from non-AUG start codons. Both compounds increase translation of a natural upstream open reading frame previously shown to initiate translation at a UUG. The compounds were also found to exacerbate increased use of UUG as a start codon (Sui(-) phenotype) conferred by haploinsufficiency of wild-type eukaryotic initiation factor (eIF) 1, or by mutation in eIF1. Furthermore, the effects of the compounds are suppressed by overexpressing eIF1, which is known to restore the fidelity of start codon selection in strains harboring Sui(-) mutations in various other initiation factors. Together, these data strongly suggest that the compounds affect the translational machinery itself to reduce the accuracy of selecting AUG as the start codon.     

Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex scans the mRNA in search of an AUG codon at which to begin translation. Start codon recognition halts scanning and triggers a number of events that commit the complex to beginning translation at that point on the mRNA. Previous studies in vitro and in vivo have indicated that eukaryotic initiation factors (eIFs) 1, 2 and 5 play key roles in these events. In addition, it was reported recently that the C-terminal domain of eIF1A is involved in maintaining the fidelity of start codon recognition. The molecular mechanisms by which these factors work together to ensure fidelity of start site selection remain poorly understood. Here, we report the quantitative characterization of energetic interactions between eIF1A, eIF5 and the AUG codon in an in vitro reconstituted yeast translation initiation system. Our results show that recognition of an AUG codon by the 43 S complex triggers an interaction between eIF5 and eIF1A, resulting in a shift in the equilibrium between two states of the pre-initiation complex. This AUG-dependent change may be a reorganization from a scanning-competent state to a scanning-incompetent state. Mutations in both eIF1A and eIF5 that increase initiation at non-AUG codons in vivo weaken the interaction between the two factors upon AUG recognition, while specifically strengthening it in response to a UUG codon. These data suggest strongly that the interaction between eIF1A and eIF5 is involved in maintaining the fidelity of start codon recognition in vivo.     

Evidence for involvement of trans-acting factors in selection of the AUG start codon during eukaryotic translational initiation.

The molecular mechanism with which an appropriate AUG codon is selected as the start site for translational initiation by eukaryotic ribosomes is not known. By using a cell-free translation system, small RNA molecules containing single AUG codons, surrounded by various nucleotide sequences, were tested for their abilities to interfere with the translation of a reporter mRNA. RNAs containing the AUG in an ACCAUGG context (Kozak consensus sequence) were able to inhibit translation of the reporter mRNA. In contrast, RNAs containing the AUG in a less favorable context for start site selection (for example, CAGAUGG) had no effect on the translation of the reporter mRNA. The effect mediated by the ACCAUGC-containing RNAs was not due to sequestration of ribosomal subunits or to particular structural features in these RNAs. To identify potential trans-acting factors that might be preferentially bound by ACCAUGG-containing RNAs, ACCAUGG- and CAGAUGC-containing RNAs with a single 4-thiouridine residue at the AUG were incubated with partially fractionated extracts, and AUG-binding proteins were identified after irradiation of the complexes with UV light and subsequent analysis by gel electrophoresis. The analysis (of such complexes in competition experiments revealed that proteins, approximately 50 and 100 kDa in size, were found to bind directly at the AUG codon embedded in the ACCAUGG motif. One of these proteins has been identified as the La autoantigen. These findings indicate that trans-acting factors may play a role in AUG start site selection during translational initiation.     

Translation of the Saccharomyces cerevisiae tcm1 gene in the absence of a 5'-untranslated leader.

The role of eukaryotic 5'-untranslated messenger RNA leaders is not entirely clear, since they share little sequence similarity among each other. The importance of the leader in determining the efficiency of translation initiation was addressed here by examining the polyribosome distribution of several leader-deletion alleles of the yeast tcm1 gene (coding for ribosomal protein L3). Shortening of this 22-nucleotide leader, or complete removal of it (the first nucleotide of the mRNA becoming the A of the translation initiation codon AUG) permitted translation, albeit reduced. Further deletion of as few as the first two nucleotides of the initiation codon leads to a substantial reduction in ribosome loading, which is compatible with inefficient initiation at the next downstream, out-of-frame, AUG triplet. A second measure of translation initiation was obtained by assaying qualitatively for the production of biologically active L3 protein using growth-resistance to trichodermin. This experiment indicates that ribosomes can recognize the correct initiation codon even in the complete absence of a leader. We conclude that the 5'-untranslated leader of the yeast tcm1 gene is not essential for accurate translation initiation, but enhances its efficiency.     

A small stem loop element directs internal initiation of the URE2 internal ribosome entry site in Saccharomyces cerevisiae

Internal initiation of translation is the process of beginning protein synthesis independent of the m(7)G cap structure at the 5'-end of an mRNA molecule. We have previously shown that the URE2 mRNA in the yeast Saccharomyces cerevisiae contains an internal ribosome entry site (IRES) whose activity is suppressed by eukaryotic initiation factor 2A (eIF2A; YGR054W). In this study, the minimal sequence required to efficiently direct internal initiation was determined using a system that abrogates cap-dependent scanning of the 40 S ribosomal subunit in both wild-type and eIF2A knock-out cells. Subsequently, secondary structural elements within the minimal sequence were determined by probing with RNases T1 and V1 and the small molecule diethylpyrocarbonate. It was found that the URE2 minimal IRES comprises a 104 nucleotide A-rich stem loop element encompassing the internal AUG codon. Interestingly, the internal AUG seems to be involved in base-pairing interactions that would theoretically hamper its ability to interact with incoming initiator tRNA molecules. Furthermore, none of the truncations used to identify the minimal IRES element were capable of abrogating the suppressive effect of eIF2A. Our data provide the first insight into the RNA structural requirements of the yeast translational machinery for cap-independent initiation of protein synthesis.     

真核细胞中多重表达框mRNA的选择性翻译起始

扫描模型和遗漏扫描模型是真核生物mRNA翻译起始的两种主要机制,但其仍存在某些例外情况,如对具有多顺反子结构的mRNA,选择性翻译起始的发生机制目前仍不清楚.本研究基于GFP蛋白开放表达框(ORF)构建了一系列重组表达载体,用以转录在移码翻译顺序及同一翻译顺序下,AUG起始密码子处于不同序列背景,以及间隔不同距离的多顺反子结构mRNA.通过转染人Bel 7402细胞系,研究了这些多顺反子结构mRNA的翻译起始模式.结果表明,在移码翻译顺序下,多顺反子mRNA可翻译出对应的不同蛋白质,而在同一翻译顺序下,GFP蛋白表达框中的多个AUG密码子,仅有首位起始密码子可发挥作用,提示核糖体在从首位起始密码子开始翻译的同时,可能会有部分核糖体继续向下扫描并识别下游的起始密码子,而这种选择性的翻译起始效率,主要取决于密码子所处的序列背景及间隔距离等因素.     

Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs.

The nucleotide sequence of the translation initiation regions of 96 Saccharomyces cerevisiae mRNAs was compiled and compared. The entire 5' untranslated sequence of most mRNAs is very rich in A-residues. G-residues are underrepresented in the untranslated region. The AUG startcodon context appeared to be distinctly different from that of animal mRNAs, although an A-residue at -3 also occurs very frequently (81 percent) in yeast mRNAs. The prevailing codon 3' adjacent to the AUG is the UCU serine codon. All these features are more extreme in the highly expressed genes. Fifty percent of all highly expressed genes use the UCU serine codon as second triplet. In this group G-residues are completely absent in the 7 bases preceding the startcodon and an A-residue occurs at position -1 and -3 at a frequency of 89 percent and 100 percent, respectively. The abundance of A-residues throughout the leader suggests that unstructured mRNA is required for efficient translation initiation in yeast. The consensus sequence for the AUG context in highly expressed genes can be summarized as follows: (Sequence: see text).     

Efficiency of translation initiation by non-AUG codons in Saccharomyces cerevisiae.

The quantitative levels of initiation of protein synthesis at codons other than AUG were determined with a CYC7-lacZ fused gene in the yeast Saccharomyces cerevisiae. AUG was the only codon which efficiently initiated translation, although some non-AUG codons allowed initiation at very low efficiency, below 1% of the normal level. Since translation initiates at codons other than AUG in at least two wild-type genes from eucaryotes, other factors presumably play a role in enhancing the activity of non-AUG codons.     

Binding of wheat germ ribosomes to bisulfite-modified reovirus messenger RNA: Evidence for a scanning mechanism

A scanning mechanism has been proposed (Kozak, 1978) to explain how eukaryotic ribosomes select the correct AUG codon for initiation of protein synthesis. The hypothesis is that a 40 S ribosomal subunit binds initially at or near the 5′-terminus of a message and subsequently migrates toward the interior of the messenger RNA, stopping when it encounters the first AUG codon, at which point a 60 S subunit joins and peptide bond formation begins. The scanning mechanism predicts that if a message were modified by introduction of a new AUG triplet upstream of the existing initiator codon, the adventitious AUG should be the preferred site for formation of an 80 S initiation complex. This prediction has been confirmed in the present studies with two reovirus messenger RNAs, in which sodium bisulfite was used to convert an ACG sequence (located in the 5′ untranslated region of each message) to AUG. Analysis of the ribosome-protected mRNA fragments recovered from sparsomycin-blocked 80 S initiation complexes revealed that a high percentage of wheat germ ribosomes were centered around the “unnatural” 5′-proximal AUG created by the bisulfite treatment, although some ribosomes were also positioned at the second (normal) initiator codon. The bisulfite modification was carried out in 7 m-urea at 37 °C. resulting in quantitative conversion of cytosine to uracil. Thus, both the primary and secondary structure of the message were drastically altered. These perturbations did not impair the efficiency of ribosome binding, nor did the highly unfolded state of the mRNA permit ribosomes to attach to spurious sites in the interior of the message. The data support a mechanism in which the initiator codon is selected by virtue of its position in a message (i.e. closest to the 5′-terminus), without regard to either the primary or secondary structure of the flanking regions.