Translation Initiation in Yeast: a Genetic and Mutational Analysis

Donahue, Thomas F.; Cigan, A. Mark; Castilho, Beatriz; Yoon, Heejong

doi:10.1007/978-3-642-73139-6_29

Cited by 7 publications

(8 citation statements)

References 16 publications

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“…The data presented here suggest that two 30-to 40-bp segments containing URFs 1 and 4, respectively, are sufficient for a pattern of translational control typical of wild-type GCN4 mRNA. The 3' proximal segment supplies the negative regulatory function required to maintain repression of GCN4 expression in nonstarvation conditions. The 5' proximal segment provides the positive regulatory function needed to overcome the inhibitory effect of the 3' proximal sequences in starvation conditions.…”

Section: Discussionmentioning

confidence: 99%

“…AUG codons 3 and 4 (counting from the 5' end) are both necessary and sufficient for efficient repression of GCN4 expression in nonstarvation conditions. After removal of AUG codons 1 and 2, these sequences repress GCN4 expression in the absence of the GCD gene products normally required for repression of the wild-type GCN4 gene.…”

mentioning

confidence: 99%

“…Just as in mammalian transcripts, upstream AUG codons occur infrequently in yeast mRNA and their insertion into the leader of a transcript generally leads to reduced translation of downstream protein-coding sequences (1,3,9,11,12,14,15,20,24). The scanning model for translation initiation accounts for this effect by postulating preferential initiation at 5' proximal AUG codons coupled with inefficient reinitiation at downstream AUG codons (10,12,24).…”

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confidence: 99%

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The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

Williams¹,

Mueller²,

Hinnebusch³

1988

Mol. Cell. Biol.

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Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRPI. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

Williams¹,

Mueller²,

Hinnebusch³

1988

Mol. Cell. Biol.

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“…Utilization of internal AUG codons as translation initiation sites appears to be very inefficient in S. cerevisiae (1,26). This fact is consistent with the idea that a preinitiation complex is assembled at the 5' end of the mRNA, scans in the 3' direction, and selects the first AUG codon suitable for initiation.…”

Section: Discussionmentioning

confidence: 99%

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence.

et al. 1988

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The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is -30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest that most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URFl-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions. The GCN4 protein of Saccharomyces cerevisiae stimulates the transcription of genes encoding amino acid biosynthetic enzymes in response to starvation for any amino acid (for a review, see reference 7). Activation of gene expression occurs as the result of increased synthesis of GCN4 under starvation conditions. Regulation of GCN4 expression in response to amino acid availability is mediated by positive (GCN) and negative (GCD) trans-acting factors (4,6,8). Each of four AUG codons present in the leader of GCN4 mRNA initiates a short open reading frame of only two or three codons before an in-frame termination codon is reached (5, 27). Removal of all four upstream AUG codons by deletion or point mutations leads to constitutive derepression of GCN4, independent of the GCN and GCD gene products that normally regulate its expression. The AUG mutations have no effect on the steady-state level of GCN4 mRNA. These results indicate that the upstream AUG codons mediate translational control of GCN4 expression by GCN and GCD regulatory factors (20,21,2...

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“…Sequences rich in G or sequences which can form double-stranded structures inhibit translation initiation probably by interfering with the scanning process [ 5 , 6, 25, 261. S. cerevisiae differs from higher eukaryotes in that the sequences flanking the initiator AUG are not very important for recognition by the S. cerevisiae ribosome [4][5][6]241. An A in the -3 position (relative to the A of the AUG codon) stimulates initiation only 2-3-fold compared to an initiator codon with U in the -3 position.…”

Section: Initiationmentioning

confidence: 99%

Translation and regulation of translation in the yeast Saccharomyces cerevisiae

Müller

Trachsel

1990

European Journal of Biochemistry

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In recent years the yeast Saccharomyces cerevisiae has become a model system for studies of eukaryotic translation and translation regulation. Analysis of mRNA structure, translation initiation factor sequences and the translation initiation pathway indicate, that translation in S. cerevisiae is very similar to translation in higher eukaryotes. The availability of powerful genetic techniques lead to the dissection in yeast of individual steps in the translation pathway, the detection of biochemical interactions between components involved in translation and the unravelling of complex regulation phenomena.

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Translation Initiation in Yeast: a Genetic and Mutational Analysis

Cited by 7 publications

References 16 publications

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence.

Translation and regulation of translation in the yeast Saccharomyces cerevisiae

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