Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control

Translational control of the GCN4 gene involves two short open reading frames in the mRNA leader (uORF1 and uORF4) that differ greatly in the ability to allow reinitiation at GCN4 following their own translation. The low efficiency of reinitiation characteristic of uORF4 can be reconstituted in a hybrid element in which the last codon of uORF1 and 10 nucleotides 3' to its stop codon (the termination region) are substituted with the corresponding nucleotides from uORF4. To define the features of these 13 nucleotides that determine their effects on reinitiation, we separately randomized the sequence of the third codon and termination region of the uORF1-uORF4 hybrid and selected mutant alleles with the high-level reinitiation that is characteristic of uORF1. The results indicate that many different A+U-rich triplets present at the third codon of uORF1 can overcome the inhibitory effect of the termination region derived from uORF4 on the efficiency of reinitiation at GCN4. Efficient reinitiation is not associated with codons specifying a particular amino acid or isoacceptor tRNA. Similarly, we found that a diverse collection of A+U-rich sequences present in the termination region of uORF1 could restore efficient reinitiation at GCN4 in the presence of the third codon derived from uORF4. To explain these results, we propose that reinitiation can be impaired by stable base pairing between nucleotides flanking the uORF1 stop codon and either the tRNA which pairs with the third codon, the rRNA, or sequences located elsewhere in GCN4 mRNA. We suggest that these interactions delay the resumption of scanning following peptide chain termination at the uORF and thereby lead to ribosome dissociation from the mRNA.

Download Full-text

Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control.

Molecular and Cellular Biology ◽

10.1128/mcb.11.1.486 ◽

1991 ◽

Vol 11 (1) ◽

pp. 486-496 ◽

Cited By ~ 131

Author(s):

J P Abastado ◽

P F Miller ◽

B M Jackson ◽

A G Hinnebusch

Keyword(s):

Amino Acid ◽

Translational Control ◽

Inhibitory Effect ◽

Open Reading Frames ◽

Start Codon ◽

Wild Type ◽

Substantial Fraction ◽

Upstream Open Reading Frames ◽

Reading Frames ◽

Do So

GCN4 encodes a transcriptional activator of amino acid-biosynthetic genes in Saccharomyces cerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in its mRNA leader. uORF4 (counting from the 5' end) is sufficient to repress GCN4 under nonstarvation conditions; uORF1 is required to overcome the inhibitory effect of uORF4 and stimulate GCN4 translation in amino acid-starved cells. Insertions of sequences with the potential to form secondary structure around uORF4 abolish derepression, indicating that ribosomes reach GCN4 by traversing uORF4 sequences rather than by binding internally to the GCN4 start site. By showing that wild-type regulation occurred even when uORF4 was elongated to overlap GCN4 by 130 nucleotides, we provide strong evidence that those ribosomes which translate GCN4 do so by ignoring the uORF4 AUG start codon. This conclusion is in accord with the fact that translation of a uORF4-lacZ fusion was lower in a derepressed gcd1 mutant than in a nonderepressible gcn2 strain. We also show that increasing the distance between uORF1 and uORF4 to the wild-type spacing that separates uORF1 from GCN4 specifically impaired the ability of uORF1 to derepress GCN4 translation. As expected, this alteration led to increased uORF4-lacZ translation in gcd1 cells. Our results suggest that under starvation conditions, a substantial fraction of ribosomes that translate uORF1 fail to reassemble the factors needed for reinitiation by the time they scan to uORF4, but become competent to reinitiate after scanning the additional sequences to GCN4. Under nonstarvation conditions, ribosomes would recover more rapidly from uORF1 translation, causing them all to reinitiate at uORF4 rather than at GCN4.

Download Full-text

Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control

Molecular and Cellular Biology ◽

10.1128/mcb.11.1.486-496.1991 ◽

1991 ◽

Vol 11 (1) ◽

pp. 486-496

Author(s):

J P Abastado ◽

P F Miller ◽

B M Jackson ◽

A G Hinnebusch

Keyword(s):

Amino Acid ◽

Translational Control ◽

Inhibitory Effect ◽

Open Reading Frames ◽

Start Codon ◽

Wild Type ◽

Substantial Fraction ◽

Upstream Open Reading Frames ◽

Reading Frames ◽

Do So

GCN4 encodes a transcriptional activator of amino acid-biosynthetic genes in Saccharomyces cerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in its mRNA leader. uORF4 (counting from the 5' end) is sufficient to repress GCN4 under nonstarvation conditions; uORF1 is required to overcome the inhibitory effect of uORF4 and stimulate GCN4 translation in amino acid-starved cells. Insertions of sequences with the potential to form secondary structure around uORF4 abolish derepression, indicating that ribosomes reach GCN4 by traversing uORF4 sequences rather than by binding internally to the GCN4 start site. By showing that wild-type regulation occurred even when uORF4 was elongated to overlap GCN4 by 130 nucleotides, we provide strong evidence that those ribosomes which translate GCN4 do so by ignoring the uORF4 AUG start codon. This conclusion is in accord with the fact that translation of a uORF4-lacZ fusion was lower in a derepressed gcd1 mutant than in a nonderepressible gcn2 strain. We also show that increasing the distance between uORF1 and uORF4 to the wild-type spacing that separates uORF1 from GCN4 specifically impaired the ability of uORF1 to derepress GCN4 translation. As expected, this alteration led to increased uORF4-lacZ translation in gcd1 cells. Our results suggest that under starvation conditions, a substantial fraction of ribosomes that translate uORF1 fail to reassemble the factors needed for reinitiation by the time they scan to uORF4, but become competent to reinitiate after scanning the additional sequences to GCN4. Under nonstarvation conditions, ribosomes would recover more rapidly from uORF1 translation, causing them all to reinitiate at uORF4 rather than at GCN4.

Download Full-text

Faculty Opinions recommendation of Translational control via protein-regulated upstream open reading frames.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.11312956.12315054 ◽

2011 ◽

Author(s):

Craig Smibert ◽

Alexander J Marsolais

Keyword(s):

Translational Control ◽

Open Reading Frames ◽

Upstream Open Reading Frames ◽

Reading Frames

Download Full-text

Heterochromatic Stellate Gene Cluster in Drosophila melanogaster: Structure and Molecular Evolution

Genetics ◽

10.1093/genetics/146.1.253 ◽

1997 ◽

Vol 146 (1) ◽

pp. 253-262 ◽

Cited By ~ 2

Author(s):

Alexei V Tulin ◽

Galina L Kogan ◽

Dominik Filipp ◽

Maria D Balakireva ◽

Vladimir A Gvozdev

Keyword(s):

Drosophila Melanogaster ◽

Molecular Evolution ◽

Protein Kinase Ck2 ◽

Unknown Origin ◽

Open Reading Frames ◽

Β Subunit ◽

Retrotransposon Insertion ◽

High Level ◽

Kinase Ck2 ◽

Reading Frames

The 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes was localized in the distal heterochromatin of the X chromosome. Of 10 sequenced genes, nine contain undamaged open reading frames with extensive similarity to protein kinase CK2 β-subunit; one gene is interrupted by an insertion. The heterochromatic array of Stellate repeats is divided into three regions by a 4.5-kb DNA segment of unknown origin and a retrotransposon insertion: the A region (∼14 Stellate genes), the adjacent B region (approximately three Stellate genes), and the C region (about four Stellate genes). The sequencing of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification. The lowest level of divergence was detected in nearby Stellate repeats. The marginal copies of the A region, truncated or interrupted by an insertion, escaped homogenization and demonstrated high levels of divergence. Comparison of copies in the B and C regions, which are separated by a retrotransposon insertion, revealed a high level of diversification. These observations suggest that homogenization takes place in the Stellate cluster, but that inserted sequences may impede this process.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3827-3836.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3827-3836

Author(s):

N P Williams ◽

P P Mueller ◽

A G Hinnebusch

Keyword(s):

Translational Control ◽

Normal Growth ◽

Regulatory Elements ◽

Growth Conditions ◽

Open Reading Frames ◽

Regulatory Function ◽

Yeast Saccharomyces Cerevisiae ◽

Reading Frame ◽

Yeast Genes ◽

Reading Frames

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 TRP1. 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.

Download Full-text

Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation

10.1101/021501 ◽

2015 ◽

Cited By ~ 2

Author(s):

David E Weinberg ◽

Premal Shah ◽

Stephen W Eichhorn ◽

Jeffrey A Hussmann ◽

Joshua B Plotkin ◽

...

Keyword(s):

Translational Control ◽

Nucleotide Composition ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Untranslated Regions ◽

Coding Regions ◽

Genome Wide ◽

Upstream Open Reading Frames ◽

Improved Methods ◽

Reading Frames

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5′- untranslated regions. Collectively, our results reveal key features of translational control in yeast and provide a framework for executing and interpreting ribosome- profiling studies.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5439-5447.1988 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5439-5447

Author(s):

P P Mueller ◽

B M Jackson ◽

P F Miller ◽

A G Hinnebusch

Keyword(s):

Translational Control ◽

Normal Growth ◽

Open Reading Frames ◽

Regulatory Function ◽

Coding Sequences ◽

Lacz Fusions ◽

Upstream Open Reading Frames ◽

Reading Frames ◽

Starvation Conditions

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 approximately 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 the 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 URF1-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.

Download Full-text

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4795-4806.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4795-4806

Author(s):

J W Xuan ◽

P Fournier ◽

N Declerck ◽

M Chasles ◽

C Gaillardin

Keyword(s):

Yarrowia Lipolytica ◽

Stop Codon ◽

Open Reading Frames ◽

Yeast Cells ◽

Lacz Gene ◽

Galactosidase Activity ◽

Phenotypic Effect ◽

Frame Fusion ◽

Beta Galactosidase ◽

Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

Download Full-text