Affecting gene expression by altering the length and sequence of the 5' leader.

Johansen, Hanne; Schümperli, Daniel; Rosenberg, Martin

doi:10.1073/pnas.81.24.7698

Cited by 87 publications

(67 citation statements)

References 23 publications

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“…3). This type of upstream reading frame, when introduced into the leader region of the bacterial galK gene, has been shown to reduce expression significantly after transfection of eucaryotic cells (11). Furthermore, the removal of all four upstream short reading frames (preceded by consensus AUGs) in the leader of the yeast GCN4 gene dramatically increased expression of this gene (34).…”

Section: Resultsmentioning

confidence: 99%

“…The fact that several mRNAs (including ASV) contain optimal AUGs upstream of the major reading frame initially seemed paradoxical. It now is clear that in some cases upstream AUGs can actually be recognized as initiator codons and that downstream reading frames may then be recognized through reinitiation (10,11,18,21,27). Furthermore, upstream AUGs, whether followed by in-frame termination codons (11) or not (18,21), can act as barriers of downstream initiation; the most efficient barriers are those which contain an AUG in the optimal initiation context.…”

Section: Resultsmentioning

confidence: 99%

“…In particular, it remains unclear whether the primary sequence or secondary structure (or both) of the mRNA leader is important in modulating mRNA translational efficiency. One approach to this question has been to alter the mRNA leader region of cloned genes which have been engineered into highefficiency expression vectors (11,18,21,23). The effect of manipulating the leader region can be measured by detection of the gene product after transfection of recipient cells.…”

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confidence: 99%

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Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.

Katz¹,

Cullen²,

Malavarca³

et al. 1986

Mol. Cell. Biol.

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The scanning hypothesis states that the initiation of translation of eucaryotic mRNAs involves binding of the 40S ribosomal subunit to the mRNA 5' terminus followed by migration along the RNA until an initiator AUG codon is encountered (i4). In the vast majority of eucaryotic mRNAs, the 40S subunit mnigrates to the 5'-proximal initiator AUG codon where translation begins. Initiator AUG codons are preceded by the consensus sequence GXXAUG; the only highly conserved nucleotide is a purine at position -3 from the AUG codon (15, 16). Initiator AUG codons which align with the major translational reading frame of mRNAs have been found between 3 and 572 nucleotides from the 5' terminus (16). A small percentage of cellular mRNAs and some viral mRNAs contain additional AUG codons which lie upstream of the major open reading frame encoded by the mRNA. These upstream AUGs have two major characteristics: (i) they are not usually preceded by consensus AUG initiation sequences, and (ii) they are followed closely by in-frame termination codons which precede the AUG start codon of the major protein reading frame. the riRNA, as has been suggested in the cases of both poliovirus (25) and ASV (5).Although several predictions of the scanning model of translation initiation have been substantiated by experimental data, several problems remain unresolved. In particular, it remains unclear whether the primary sequence or secondary structure (or both) of the mRNA leader is important in modulating mRNA translational efficiency. One approach to this question has been to alter the mRNA leader region of cloned genes which have been engineered into highefficiency expression vectors (11,18,21,23). The effect of manipulating the leader region can be measured by detection of the gene product after transfection of recipient cells. This approach has been useful in demonstrating that upstream initiator AUG codons can interfere with the initiation of downstream reading frames. However, this method is not ideal for determining the normal function of the 5' leader region for at least two reasons: (i) the 5' terminus of the mRNA is donated from the expression vector, thus generating a chimeric, artificial mRNA, and (ii) the host cell which has been engineered for high expression is not the normal environment for the expressed RNA.The model system we have chosen to examine the effect of the leader region on gene expression utilizes the ASV envelope (env) gene and offers several advantages. The transcription of env mRNA is directed by the natural retroviral long terminal repeat (LTR) promoter, thus generating a native mRNA. The assay is also performed using quail cells, a natural host for this virus. The most important advantage, however, is the existence of sensitive, quantitative assays for both env mRNA and env protein (2-4). To examine the effect of the viral leader on env mRNA expression, we have introduced both natural leader segments from related avian retroviruses and artificially generated leader deletion muta-372 on May 12, 2018 by guest

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.

Katz¹,

Cullen²,

Malavarca³

et al. 1986

Mol. Cell. Biol.

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show abstract

“…6 for a review). The use of galK as a selective marker has also been adapted to develop gene fusion vectors to study transcriptional regulatory elements in the gram-positive bacteria Streptomyces (7), yeast (8)(9)(10)(11) and higher cell systems (5,(12)(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Structure of the galactokinase gene ofEscherichia coli, the last (?) gene of thegaloperon

et al. 1985

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We present the nucleot'de sequence of the galactokinase gene (galK) of Escherichia coli including its 5' and 3' flanking regions. This DNA sequence derives from the Xgal8 transducing phage and is identical to the sequence present in the galK gene fusion vectors, pKO and pKG, commonly used to study transcriptional regulatory elements. We define the precise 3' junction between the bacterial and phage sequences in Xgal8 and demonstrate that this junction probably results from a homologous recombination event between identical 9 bp sequences common to the gal operon and phage X. Moreover, we examine the 300 bp region located immediately beyond galK for transcription termination function and find no gal operon terminator. Lastly, we compare the gaIK genes of E. coli and the yeast S. cerevisiae and find several regions of strong homology among which is a potential ATP-binding site homology shared by a variety of ATP-binding proteins including protein kinases encoded by mammalian oncogenes.

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“…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).…”

mentioning

confidence: 99%

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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Affecting gene expression by altering the length and sequence of the 5' leader.

Cited by 87 publications

References 23 publications

Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.

Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.

Structure of the galactokinase gene ofEscherichia coli, the last (?) gene of thegaloperon

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

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