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The gateway pDEST17 expression vector encodes a -1 ribosomal frameshifting sequence.

Belfield EJ, Hughes RK, Tsesmetzis N, Naldrett MJ, Casey R - Nucleic Acids Res. (2007)

Bottom Line: We demonstrate unambiguously the frameshift through a combination of Edman degradation, MALDI-ToF mass fingerprint analysis of tryptic peptides and MALDI-ToF reflectron in-source decay (rISD) sequencing.The degree of frameshifting depends on the nature of the sequence being expressed and ranged from 25 to 60%.These findings suggest that caution should be exercised when employing pDEST17 for high-level protein expression and that the attB1 site has some potential as a tool for studying -1 frameshifting.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. eric.belfield@bbsrc.ac.uk

ABSTRACT
The attB1 site in the Gateway (Invitrogen) bacterial expression vector pDEST17, necessary for in vitro site-specific recombination, contains the sequence AAA-AAA. The sequence A-AAA-AAG within the Escherichia coli dnaX gene is recognized as 'slippery' and promotes -1 translational frameshifting. We show here, by expressing in E. coli several plant cDNAs with and without single nucleotide deletions close to the translation initiation codons, that pDEST17 is intrinsically susceptible to -1 ribosomal frameshifting at the sequence C-AAA-AAA. The deletion mutants produce correct-sized, active enzymes with a good correlation between enzyme amount and activity. We demonstrate unambiguously the frameshift through a combination of Edman degradation, MALDI-ToF mass fingerprint analysis of tryptic peptides and MALDI-ToF reflectron in-source decay (rISD) sequencing. The degree of frameshifting depends on the nature of the sequence being expressed and ranged from 25 to 60%. These findings suggest that caution should be exercised when employing pDEST17 for high-level protein expression and that the attB1 site has some potential as a tool for studying -1 frameshifting.

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Sequence and predicted secondary structures of the MtHPLF, AtAOS and PsLOX3-FS RNAs and putative −1 frameshift-inducing site. The frameshift slippery sequence is shown in bold. All nucleotides are Watson–Crick base paired except the U:G wobble marked with a star in the PsLOX3-FS RNA structure. The stem-loop structures were predicted using the mfold software (38). Thermodynamic free energy (ΔG) was calculated at 37°C and 1 M Na+ concentration. The arrows next to the MtHPLF-FS RNA structure indicate the nucleotides mutated to study the possible consequences of changes in the thermodynamic stability of the stem loop on −1 frameshifting.
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Figure 5: Sequence and predicted secondary structures of the MtHPLF, AtAOS and PsLOX3-FS RNAs and putative −1 frameshift-inducing site. The frameshift slippery sequence is shown in bold. All nucleotides are Watson–Crick base paired except the U:G wobble marked with a star in the PsLOX3-FS RNA structure. The stem-loop structures were predicted using the mfold software (38). Thermodynamic free energy (ΔG) was calculated at 37°C and 1 M Na+ concentration. The arrows next to the MtHPLF-FS RNA structure indicate the nucleotides mutated to study the possible consequences of changes in the thermodynamic stability of the stem loop on −1 frameshifting.

Mentions: Frameshifting rates are generally dependent on a number of stimulatory sequences, including a downstream hairpin or pseudoknot that causes slowing, or pausing, of the ribosome long enough at the slippery sequence for frameshifting to occur (34,35) and an upstream Shine–Dalgarno sequence that pairs with the 16S RNA, causing ribosomal ‘stress’ (1). Our analysis of the sequence proximal to the pDEST17 slippery site found no potential Shine–Dalgarno sequences. Secondary structure analysis for stimulatory frameshifting sequences such as hairpin loops (26), or pseudoknots (36) (Figure 5) of the highly frameshifting MtHPLF-FS revealed that the RNA fold with the lowest minimum free energy had a potential stem-loop of five predicted Watson–Crick base pairs (mfold (37)). This hairpin is thermodynamically more stable and compact than those predicted in the RNAs for the FS PsLOX3 and AtAOS stem-loop structures, which have a maximum of two and four consecutive paired nucleotides, respectively and may explain why lower frameshift rates (of 36 and 25%, respectively) were observed compared to the MtHPLF-FS clone. The MtHPLF-FS secondary structure has some of the characteristics that Antao and Tinoco (38) reported; they found that hairpin tetraloops, with stem sizes of four or five bases, could form extra stable hairpins when the loop-closing base pair was A–U. If the stem-loop structures predicted in Figure 5 are real then a combination of hairpin thermodynamic stability and/or the identity of the A–U loop-closing base pair could explain why the frameshifting efficiency is higher for MtHPLF-FS RNA than the PsLOX3 and AtAOS transcripts. To test this hypothesis we modified the MtHPLF-FS RNA molecule using site-directed mutagenesis. First, we generated a single nucleotide point mutant to change the A–U loop-closing pair to a non complementary A-C pair. In addition, we modified a cytosine to a guanine that was predicted to be the third nucleotide of a five base stem of a stem-loop structure (see Figure 5). The predicted secondary structures of the mutated MtHPLF-FS RNAs were thermodynamically less stable (A–C pair: ΔG = −2.8 kcal/mol and cytosine to a guanine: ΔG = −0.1 kcal/mol) compared to the original MtHPLF-FS RNA (ΔG = −3.6 kcal/mol) (RNA secondary structures predicted by the mfold programme (37) are not shown). The specific activities of both mutants with the substrate 13-HPOT (data not shown) were not significantly different from that of the MtHPLF-FS clone even though the proposed stem loops in both mutants were predicted to be thermodynamically less stable suggesting the predicted secondary structures may not be accurate.Figure 5.


The gateway pDEST17 expression vector encodes a -1 ribosomal frameshifting sequence.

Belfield EJ, Hughes RK, Tsesmetzis N, Naldrett MJ, Casey R - Nucleic Acids Res. (2007)

Sequence and predicted secondary structures of the MtHPLF, AtAOS and PsLOX3-FS RNAs and putative −1 frameshift-inducing site. The frameshift slippery sequence is shown in bold. All nucleotides are Watson–Crick base paired except the U:G wobble marked with a star in the PsLOX3-FS RNA structure. The stem-loop structures were predicted using the mfold software (38). Thermodynamic free energy (ΔG) was calculated at 37°C and 1 M Na+ concentration. The arrows next to the MtHPLF-FS RNA structure indicate the nucleotides mutated to study the possible consequences of changes in the thermodynamic stability of the stem loop on −1 frameshifting.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851650&req=5

Figure 5: Sequence and predicted secondary structures of the MtHPLF, AtAOS and PsLOX3-FS RNAs and putative −1 frameshift-inducing site. The frameshift slippery sequence is shown in bold. All nucleotides are Watson–Crick base paired except the U:G wobble marked with a star in the PsLOX3-FS RNA structure. The stem-loop structures were predicted using the mfold software (38). Thermodynamic free energy (ΔG) was calculated at 37°C and 1 M Na+ concentration. The arrows next to the MtHPLF-FS RNA structure indicate the nucleotides mutated to study the possible consequences of changes in the thermodynamic stability of the stem loop on −1 frameshifting.
Mentions: Frameshifting rates are generally dependent on a number of stimulatory sequences, including a downstream hairpin or pseudoknot that causes slowing, or pausing, of the ribosome long enough at the slippery sequence for frameshifting to occur (34,35) and an upstream Shine–Dalgarno sequence that pairs with the 16S RNA, causing ribosomal ‘stress’ (1). Our analysis of the sequence proximal to the pDEST17 slippery site found no potential Shine–Dalgarno sequences. Secondary structure analysis for stimulatory frameshifting sequences such as hairpin loops (26), or pseudoknots (36) (Figure 5) of the highly frameshifting MtHPLF-FS revealed that the RNA fold with the lowest minimum free energy had a potential stem-loop of five predicted Watson–Crick base pairs (mfold (37)). This hairpin is thermodynamically more stable and compact than those predicted in the RNAs for the FS PsLOX3 and AtAOS stem-loop structures, which have a maximum of two and four consecutive paired nucleotides, respectively and may explain why lower frameshift rates (of 36 and 25%, respectively) were observed compared to the MtHPLF-FS clone. The MtHPLF-FS secondary structure has some of the characteristics that Antao and Tinoco (38) reported; they found that hairpin tetraloops, with stem sizes of four or five bases, could form extra stable hairpins when the loop-closing base pair was A–U. If the stem-loop structures predicted in Figure 5 are real then a combination of hairpin thermodynamic stability and/or the identity of the A–U loop-closing base pair could explain why the frameshifting efficiency is higher for MtHPLF-FS RNA than the PsLOX3 and AtAOS transcripts. To test this hypothesis we modified the MtHPLF-FS RNA molecule using site-directed mutagenesis. First, we generated a single nucleotide point mutant to change the A–U loop-closing pair to a non complementary A-C pair. In addition, we modified a cytosine to a guanine that was predicted to be the third nucleotide of a five base stem of a stem-loop structure (see Figure 5). The predicted secondary structures of the mutated MtHPLF-FS RNAs were thermodynamically less stable (A–C pair: ΔG = −2.8 kcal/mol and cytosine to a guanine: ΔG = −0.1 kcal/mol) compared to the original MtHPLF-FS RNA (ΔG = −3.6 kcal/mol) (RNA secondary structures predicted by the mfold programme (37) are not shown). The specific activities of both mutants with the substrate 13-HPOT (data not shown) were not significantly different from that of the MtHPLF-FS clone even though the proposed stem loops in both mutants were predicted to be thermodynamically less stable suggesting the predicted secondary structures may not be accurate.Figure 5.

Bottom Line: We demonstrate unambiguously the frameshift through a combination of Edman degradation, MALDI-ToF mass fingerprint analysis of tryptic peptides and MALDI-ToF reflectron in-source decay (rISD) sequencing.The degree of frameshifting depends on the nature of the sequence being expressed and ranged from 25 to 60%.These findings suggest that caution should be exercised when employing pDEST17 for high-level protein expression and that the attB1 site has some potential as a tool for studying -1 frameshifting.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. eric.belfield@bbsrc.ac.uk

ABSTRACT
The attB1 site in the Gateway (Invitrogen) bacterial expression vector pDEST17, necessary for in vitro site-specific recombination, contains the sequence AAA-AAA. The sequence A-AAA-AAG within the Escherichia coli dnaX gene is recognized as 'slippery' and promotes -1 translational frameshifting. We show here, by expressing in E. coli several plant cDNAs with and without single nucleotide deletions close to the translation initiation codons, that pDEST17 is intrinsically susceptible to -1 ribosomal frameshifting at the sequence C-AAA-AAA. The deletion mutants produce correct-sized, active enzymes with a good correlation between enzyme amount and activity. We demonstrate unambiguously the frameshift through a combination of Edman degradation, MALDI-ToF mass fingerprint analysis of tryptic peptides and MALDI-ToF reflectron in-source decay (rISD) sequencing. The degree of frameshifting depends on the nature of the sequence being expressed and ranged from 25 to 60%. These findings suggest that caution should be exercised when employing pDEST17 for high-level protein expression and that the attB1 site has some potential as a tool for studying -1 frameshifting.

Show MeSH
Related in: MedlinePlus