Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel

Lu, Jianjun; Kobertz, William R.; Deutsch, Carol

doi:10.1016/j.jmb.2007.06.038

Cited by 107 publications

(142 citation statements)

References 47 publications

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“…Though several recent studies have suggested that positively charged amino acids may impede the progress of the peptide chain through the negatively charged ribosomal exit tunnel (Lu et al 2007;Lu and Deutsch 2008), we observed no consistent enrichment for codons encoding positive amino acids in corrected Ribo coverage either within or upstream of the footprints in any data sets ( Fig. 4B; Supplemental Figs.…”

Section: No Evidence That Positive Amino Acids Stall Ribosomescontrasting

confidence: 69%

“…However, this is most likely due to the remaining effects of library construction (see Supplemental Material). In addition, there is no evidence of enrichment among upstream codons (À8 to À1), as would be expected if positive amino acids slow translation as they pass through the negatively charged ribosome exit tunnel (see Supplemental Material; Lu et al 2007;Charneski and Hurst 2013). This pattern disappeared completely in both data sets when the order of codons was randomly shuffled within each gene, preserving the relative positions of mapped reads, indicating that it was not an artifact of the relationship between codon order and patterns of read mapping positions within transcripts (Supplemental Fig.…”

Section: Ribosome Occupancy Is Associated With Proline Codonsmentioning

confidence: 95%

“…Other factors thought to slow translation rates include the presence of mRNA secondary structure, which must be ''unwound'' by ribosomes (Namy et al 2006;Wen et al 2008); wobble base-pairing, which can introduce nonoptimal geometries in codon-anticodon interactions (Thomas et al 1988;Kato et al 1990); codons encoding positively charged amino acids, which may participate in electrostatic interactions with the negatively charged ribosomal exit tunnel (Lu et al 2007;Lu and Deutsch 2008;Tuller et al 2011;Charneski and Hurst 2013); and proline, which is inefficiently incorporated into polypeptides due to the unique structure of its imino side-chain (Muto and Ito 2008;Wohlgemuth et al 2008;Pavlov et al 2009;Johansson et al 2011;Doerfel et al 2013;Gutierrez et al 2013;Ude et al 2013;Zinshteyn and Gilbert 2013). Interpretation of the relative contributions of these factors has been challenging, as their effects have typically been studied in conditions not normally encountered in living cells-such as within genes with low CUB but extremely high mRNA levels (Gingold and Pilpel 2011;Plotkin and Kudla 2011).…”

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confidence: 99%

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Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation

2014

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The recent advent of ribosome profiling-sequencing of short ribosome-bound fragments of mRNA-has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling data set have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a method that implicated positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that it produces false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling.

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Section: No Evidence That Positive Amino Acids Stall Ribosomescontrasting

confidence: 69%

Section: Ribosome Occupancy Is Associated With Proline Codonsmentioning

confidence: 95%

mentioning

confidence: 99%

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Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation

2014

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“…Compared with model reactions between positively charged maleimides and thiols, the modification of E1 T14C by CTX-Clv is Ϸ1,000-fold faster (19). This faster modification rate was expected on the basis of previous affinity-driven modifying agents, which increase the reagent's effective molarity, thereby accelerating the chemical reaction (20,21).…”

Section: Resultsmentioning

confidence: 94%

“…4a eliminate stoichiometries with an odd number of KCNE peptides (Appendix). We first pretreated Q1/E1 T14C complexes with a membrane-impermeant maleimide (Mal-ES) (19) to randomly and independently react with a fraction of the E1 T14C peptides. We then determined the percentage of Q1/E1 T14C complexes that were fully modified by this pretreatment by measuring the reversibility of CTX-Clv after washout (Fig.…”

Section: Resultsmentioning

confidence: 99%

Counting membrane-embedded KCNE β-subunits in functioning K ⁺ channel complexes

Morin

Kobertz

2008

Proc. Natl. Acad. Sci. U.S.A.

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112

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Ion channels are multisubunit proteins responsible for the generation and propagation of action potentials in nerve, skeletal muscle, and heart as well as maintaining salt and water homeostasis in epithelium. The subunit composition and stoichiometry of these membrane protein complexes underlies their physiological function, as different cells pair ion-conducting ␣-subunits with specific regulatory ␤-subunits to produce complexes with diverse ion-conducting and gating properties. However, determining the number of ␣-and ␤-subunits in functioning ion channel complexes is challenging and often fraught with contradictory results. Here we describe the synthesis of a chemically releasable, irreversible K ؉ channel inhibitor and its iterative application to tally the number of ␤-subunits in a KCNQ1/KCNE1 K ؉ channel complex. Using this inhibitor in electrical recordings, we definitively show that there are two KCNE subunits in a functioning tetrameric K ؉ channel, breaking the apparent fourfold arrangement of the ion-conducting subunits. This digital determination rules out any measurable contribution from supra, sub, and multiple stoichiometries, providing a uniform structural picture to interpret KCNE ␤-subunit modulation of voltage-gated K ؉ channels and the inherited mutations that cause dysfunction. Moreover, the architectural asymmetry of the K ؉ channel complex affords a unique opportunity to therapeutically target ion channels that coassemble with KCNE ␤-subunits.membrane proteins that open and close in response to changes in membrane potential. To regulate cellular potassium flow in both excitable and nonexcitable tissues, Q1 channels coassemble with regulatory KCNE peptides, forming membraneembedded K ϩ channel complexes with various voltage-sensing and gating properties (1). The importance of forming a properly assembled Q1-KCNE complex is underscored by the mutations that give rise to long QT syndrome and congenital hearing loss (2, 3). Although the fourfold arrangement of the Q1 ␣-subunits along the ion conduction pathway is established and unquestioned, the number of KCNE ␤-subunits in the K ϩ channel complex has been a long-standing and heated debate (4-7). Goldstein and coworkers (6) have strongly argued that Q1 channels average two KCNE peptides per complex; however, they could not definitively rule out K ϩ channel complexes containing a solitary KCNE peptide. Another complicating factor is the notion that Q1 channels form complexes with KCNE ␤-subunits with multiple stoichiometries (5, 8). Thus, approaches that measure the average number of KCNE peptides in the Q1 channel complex at the cell surface are unable to readily discern between fixed and various stoichiometries.To determine the stoichiometry or stoichiometries of Q1-KCNE K ϩ channel complexes, we devised an iterative cell surface modification scheme where the labeling reagent specifically binds to the outer vestibule of the K ϩ conduction pore while covalently modifying a cysteine-bearing KCNE peptide in the channel complex (Fig. 1). Once th...

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Sucrose sensing through nascent peptide‐meditated ribosome stalling at the stop codon of Arabidopsis bZIP11 uORF2

et al. 2017

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Arabidopsis bZIP11 is a transcription factor that modulates amino acid metabolism under high-sucrose conditions. Expression of bZIP11 is downregulated in a sucrose-dependent manner during translation. Previous in vivo studies have identified the second upstream open reading frame (uORF2) as an essential regulatory element for the sucrose-dependent translational repression of bZIP11. However, it remains unclear how uORF2 represses bZIP11 expression under high-sucrose conditions. Through biochemical experiments using cell-free translation systems, we report on sucrose-mediated ribosome stalling at the stop codon of uORF2. The C-terminal 10 amino acids (29-SFSVxFLxxLYYV-41) of uORF2 are important for ribosome stalling. Our results demonstrate that uORF2 encodes a regulatory nascent peptide that functions to sense intracellular sucrose abundance. This is the first biochemical identification of the intracellular sucrose sensor.

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Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel

Cited by 107 publications

References 47 publications

Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation

Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation

Counting membrane-embedded KCNE β-subunits in functioning K ⁺ channel complexes

Sucrose sensing through nascent peptide‐meditated ribosome stalling at the stop codon of Arabidopsis bZIP11 uORF2

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Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel

Cited by 107 publications

References 47 publications

Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation

Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation

Counting membrane-embedded KCNE β-subunits in functioning K + channel complexes

Sucrose sensing through nascent peptide‐meditated ribosome stalling at the stop codon of Arabidopsis bZIP11 uORF2

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Counting membrane-embedded KCNE β-subunits in functioning K ⁺ channel complexes