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The evolutionarily conserved eukaryotic arginine attenuator peptide regulates the movement of ribosomes that have translated it - PubMed

The evolutionarily conserved eukaryotic arginine attenuator peptide regulates the movement of ribosomes that have translated it

Z Wang et al. Mol Cell Biol. 1998 Dec.

Abstract

Translation of the upstream open reading frame (uORF) in the 5' leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control.

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Figures

FIG. 1
FIG. 1

The 5′ leader regions of arg-2–LUC genes used in this study (see also Table 1). (A) Sequences of wild-type and mutant templates in which the AAP is encoded by a uORF. The sequence shown begins with the T7 RNA polymerase-binding site and ends within the LUC coding region (26). The amino acid sequences of the arg-2 AAP and the N terminus of LUC are indicated. Point mutations are shown below the wild-type sequence. The endpoints of deletion mutations that shorten the intercistronic region are indicated by the boxed nucleotides. All deletions share the 3′ endpoint, which is indicated by a horizontal arrow above the sequence. The extent of each deletion is indicated (e.g., Δ71 removes the greatest number of nucleotides, leaving 5′-CC-3′ between the uORF termination and LUC initiation codons). The sequence for which the reverse complement was synthesized and used as primer ZW4 for toeprint analysis is indicated by a horizontal arrow below the sequence. (B) Sequences of templates containing wild-type and mutant AAP-LUC fusion genes. The sequence shown begins with the T7 RNA polymerase-binding site and ends within the LUC coding region; the amino acid sequence of the N terminus of the AAP-LUC fusion polypeptide is indicated. Point mutations are shown below the wild-type sequence. The mutation indicated by ↑ improves the initiation context for uORF translation. The fs mutation is a −1 frameshift in which the first nucleotide of the Gln codon bridging the AAP and LUC coding sequences is deleted.

FIG. 2
FIG. 2

Effects of shortening the distance between the uORF termination codon and the downstream LUC initiation codon on Arg-specific regulation. Equal amounts of synthetic RNA transcripts (120 ng) were translated in 20-μl reaction mixtures for 20 min at 25°C. Reaction mixtures contained 10 μM (−) or 500 μM (+) Arg and a 10 μM concentration of each of the other 19 amino acids. The transcripts examined are indicated at the top of the lanes. After 20 min of translation, the translation mixtures were toeprinted with primer ZW4 and analyzed next to dideoxynucleotide sequences of the corresponding DNA template. The nucleotide complementary to the dideoxynucleotide added to each reaction mixture is indicated above the corresponding lane so that the sequence of each template can be directly deduced; the 5′-to-3′ sequence reads from top to bottom. The asterisks indicate the positions of premature transcription termination products corresponding to ribosomes at the uORF termination codon; brackets indicate ribosomes stalled behind those at the termination codon. The closed arrowheads indicate ribosomes at the uORF initiation codon; the open arrowheads indicate ribosomes at the LUC initiation codon. wt, wild-type.

FIG. 3
FIG. 3

Effects of terminating uORF peptide synthesis with each of the three termination codons, UAA (lanes 1 to 4), UAG (lanes 5 to 8), and UGA (lanes 9 to 12), on Arg-specific regulation. Equal amounts of synthetic RNA transcripts (120 ng) were translated in 20-μl reaction mixtures for 20 min at 25°C. Reaction mixtures contained 10 μM (−) or 500 μM (+) Arg and a 10 μM concentration of each of the other 19 amino acids; they were analyzed by toeprinting as described in the legend to Fig. 2. The RNAs are from Δ71 deletion constructs containing either the wild-type (wt) or D12N mutant uORFs terminated with UAA, UAG, or UGA codons (Fig. 1 and Table 1) as indicated at the top of the lanes. Dideoxynucleotide sequencing reactions for the pPR102 template containing the wild-type uORF terminated with UAA are shown on the left (lanes C′, T′, A′, and G′). Arrows indicate the positions of premature transcription termination products corresponding to ribosomes bound at the uORF initiation codon (AUGAAP) and the uORF termination codon (TermAAP); brackets indicate the positions of ribosomes stalled behind those at the termination codon.

FIG. 4
FIG. 4

Analyses of [35S]methionine-labeled polypeptides produced by translation of synthetic RNA transcripts in N. crassa cell extracts. Micrococcal nuclease-treated N. crassa extracts (20 μl containing 2 μCi of [35S]methionine) were programmed with 120 ng of the indicated RNAs and incubated for 30 min at 25°C. Reactions were stopped by immersing the tube in liquid nitrogen and examined by SDS-PAGE in 10% polyacrylamide gels. Radiolabeled translation products were visualized by phosphorimaging; the positions of molecular mass markers (in kilodaltons) visualized by staining with Coomassie blue are indicated on the left. Lanes: 1, reaction mixture with no added RNA; 2, with RNA encoding firefly LUC; 3, with RNA encoding the AAP-LUC fusion polypeptide in the wild-type initiation context; 4, with RNA encoding the AAP-LUC fusion polypeptide in the improved initiation context; 5, with RNA encoding the AAP coding region frameshifted (fs) with respect to the LUC coding region; 6, with RNA encoding sea pansy (sp) LUC.

FIG. 5
FIG. 5

Time courses of translation of RNAs encoding AAP-LUC, D12N AAP-LUC, and sea pansy LUC enzymes in reaction mixtures containing 10 or 500 μM Arg. Translation in reaction mixtures (120 μl) was initiated by using a mixture of RNAs: 150 ng of RNA encoding the wild-type AAP-LUC fusion and 36 ng of RNA encoding sea pansy LUC (A) or 150 ng of RNA encoding the D12N AAP-LUC fusion and 36 ng of RNA encoding sea pansy LUC (B). Reaction mixtures were incubated at 25°C and contained either 10 or 500 μM Arg and a 10 μM concentration of each of the other 19 amino acids. Firefly LUC production (in reaction mixtures containing 10 [○] or 500 [◊] μM Arg) and sea pansy LUC production (in reaction mixtures containing 10 [□] or 500 [×] μM Arg) were determined by using the dual luciferase assay as described in Materials and Methods. RLU, relative light units.

FIG. 6
FIG. 6

Time courses of translation of RNAs encoding the wild-type AAP as an N-terminal domain and as a uORF product. Reaction conditions were as described in the legend to Fig. 5. (A) Results with 150 ng of RNA encoding the wild-type AAP-LUC fusion and 36 ng of RNA encoding sea pansy LUC; (B) results with 150 ng of RNA encoding the wild-type AAP as a uORF region and LUC as a separate downstream coding region with its own initiation codon and 36 ng of RNA encoding sea pansy LUC. The inset in panel A shows the combined data for translation of the internal control sea pansy LUC RNA in the four reaction mixtures plotted in panels A and B. Firefly LUC production (in reaction mixtures containing 10 [○] or 500 [◊] μM Arg) and sea pansy LUC production were determined by using the dual luciferase assay as described in Materials and Methods. RLU, relative light units.

FIG. 7
FIG. 7

Effects of mutations on Arg-specific regulation of AAP-LUC fusion constructs. Equal amounts of synthetic RNA transcripts (120 ng) were translated in reaction mixtures and analyzed by toeprinting as described in the legend to Fig. 2. The transcripts encoded the wild-type (wt) AAP-LUC fusion or the D12N mutant AAP-LUC fusion as indicated in either the wild-type or improved (↑) initiation contexts. Dideoxynucleotide sequencing reactions for the template containing the wild-type AAP-LUC fusion are shown on the left (lanes C′, T′, A′, and G′). The products obtained from primer extension of pure AAP-LUC RNA (18 ng) in the absence of translation reaction mixture (−EXT; lane 9) and from a translation reaction mixture not programmed with RNA (−RNA; lane 10) are shown for comparison. The arrow indicates the position of the premature transcription termination products corresponding to ribosomes bound at the AAP initiation codon (AUGAAP). The arrowhead indicates the position of premature termination products corresponding to ribosomes stalled in the presence of a high level of Arg at the codon immediately following the 24 codons of the AAP. The bracket indicates the position of premature termination products corresponding to ribosomes stalled in the presence of a high level of Arg in the LUC coding region.

FIG. 8
FIG. 8

Effects of puromycin on Arg-specific regulation of AAP-LUC fusion constructs. Equal amounts of synthetic RNA transcripts (120 ng) were translated in reaction mixtures and analyzed by toeprinting as described in the legend to Fig. 2. The transcripts encoded either the D12N mutant AAP-LUC fusion or the wild-type (wt) AAP-LUC fusion as indicated. Puromycin (Pur) was added where indicated, as described in the text. Dideoxynucleotide sequencing reactions for the template containing the wild-type AAP-LUC fusion are shown on the left (lanes C′, T′, A′, and G′). The arrow indicates the position of the premature transcription termination products corresponding to ribosomes bound at the AAP initiation codon (AUGAAP). The arrowhead indicates the position of premature termination products corresponding to ribosomes stalled in the presence of a high level of Arg at the codon immediately following the 24 codons of the AAP. The bracket indicates the position of premature termination products corresponding to ribosomes stalled in the presence of a high level of Arg in the LUC coding region.

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