Conformational Dynamics of the RNA G-Quadruplex and its Effect on Translation Efficiency

Abstract

During translation, intracellular mRNA folds co-transcriptionally and must refold following the passage of ribosome. The mRNAs can be entrapped in metastable structures during these folding events. In the present study, we evaluated the conformational dynamics of the kinetically favored, metastable, and hairpin-like structure, which disturbs the thermodynamically favored G-quadruplex structure, and its effect on co-transcriptional translation in prokaryotic cells. We found that nascent mRNA forms a metastable hairpin-like structure during co-transcriptional folding instead of the G-quadruplex structure. When the translation progressed co-transcriptionally before the metastable hairpin-like structure transition to the G-quadruplex, function of the G-quadruplex as a roadblock of the ribosome was sequestered. This suggested that kinetically formed RNA structures had a dominant effect on gene expression in prokaryotes. The results of this study indicate that it is critical to consider the conformational dynamics of RNA-folding to understand the contributions of the mRNA structures in controlling gene expression.

Keywords: G-quadruplex; co-transcriptional folding; co-translational refolding; conformational dynamics; metastable structure; translation suppression.

Figure 1

G-rich sequence variants designed to form metastable hairpin-like structures with different stabilities. (a) G-rich sequence elements and reporter mRNA constructs. Guanine nucleobases expected to be involved in the formation of the G-quadruplex structure are given in red. Nucleotides mutated from the wild-type sequence are indicated in italics. The amino acid sequence is indicated below the sequence of the nucleic acids and the reporter construct is shown schematically. (b) Secondary structures of the G-rich sequence variants predicted using the Mfold program. Thermodynamic stabilities (ΔG°) of the secondary structures predicted by the Mfold program are given.

Figure 2

Co-transcriptional formation of the G-quadruplex depending on the stability of metastable hairpin-like structures. (a) mRNA transcripts of the reaction without DNA template (lane 1), with DNA template for wild-type (lane 2), mutant A (lane 3), mutant B (lane 4), and mutant C (lane 5) were electrophoresed on an agarose gel. mRNAs were stained with ethidium bromide and imaged at 532 nm excitation and 575 nm emission wavelengths. (b,c) Fluorescence intensities of NMM mixed with mRNA transcripts immediately after transcription (b) or after refolding by heating to 70 °C and then cooling to 25 °C (c). mRNA transcripts were diluted in a buffer containing 50 mM HEPES-KOH (pH 7.6), 5 mM magnesium acetate, 100 mM potassium glutamate, 2 mM spermidine, 0.01% Tween 20, 0.01% DMSO, and 500 nM NMM. NMM fluorescence was measured at 37 °C at 400 nm excitation and 615 nm emission wavelengths. Values are expressed as means ± S.D. of experiments performed in triplicates.

Figure 3

Protein expression levels from intact mRNA and after its refolding using the in vitro translation system of E. coli S30 extract. (a) Relative luminescence intensities from Renilla luciferase translated from mRNA transcripts with (red) and without (blue) refolding. Luminescence signals were measured after addition of coelenterazine (5 μM) to the translated products and were normalized to the signal obtained from mutant A mRNA. Values are expressed as mean ± S.D. of triplicate experiments. Asterisks indicate two-tailed P-values for Student’s t-test: * P < 0.05 and ** P < 0.01. (b) Effect of the mRNA structure elements formed during co-transcriptional folding or refolding of wild-type G-rich sequence on translation reaction.

Figure 4

Time course of the levels of protein expression affected by co-transcriptional and co-translational RNA conformational dynamics within the hairpin-like and G-quadruplex structures. (a) Time course of co-transcriptional translation of reporter genes encoding wild-type (blue), mutant A (pink), mutant B (green), and mutant C (purple) sequences. DNA templates were mixed with the PURE system solution in the presence of T7 RNA polymerase and incubated at 37 °C. (b) Predicted suppression of translation caused by accumulation of the mutant C mRNA, which formed the G-quadruplex, with increasing reaction time. (c) Time course of translation of the refolded mRNAs with G-rich sequence of the wild-type (blue), mutant A (pink), mutant B (green), and mutant C (purple). Transcribed mRNAs refolded at 70 °C were mixed with PURE system solution and incubated at 37 °C. (d) Schematic of co-translational folding of metastable hairpin-like structure in wild-type mRNA that allows uninhibited translation by a subsequent ribosome. In (a) and (c), luminescence intensities of Renilla luciferase are expressed as mean ± S.D. of triplicate experiments.

Figure 5

Gene expression in E. coli dominated by kinetically favored metastable mRNA structure. (a,b) Normalized luminescence intensities of the E. coli lysate cultured in the absence (a) or presence (b) of 0.5 μM tetracycline. Protein expression was induced by 100 μM β-D-1-thiogalactopyranoside in 2× YT medium containing 100 mM potassium glutamate for 1 h. Luminescence signals were normalized by adjusting to an optical density of 600 nm of E. coli cells. Values are expressed as mean ± S.D. of triplicated E. coli culturing wells. Asterisks indicate two-tailed P-values for the Student’s t-test: * P < 0.05 and ** P < 0.01. (c) Illustration of the co-transcriptional translation in usual culturing conditions of E. coli, in which the ribosome translates a region of G-rich elements before forming the G-quadruplex. (d) Illustration of co-transcriptional translation in the presence of tetracycline, in which the level of protein expression is dominated by kinetically favored mRNA structures. Only mRNA of mutant C, which forms the G-quadruplex by bypassing any metastable structure, reduces the level of protein expression due to the roadblock function of the G-quadruplex.