从Stanley Miller的放电实验到“RNA世界”的提出，生命起源研究越来越受到科学界的重视。近20年来在Science和Nature上发表的相关文章数以百计，而Miller的放电实验到RNA世界等生命起源的概念在学术界已经众所周知了。今年年初，常年研究生命起源的Breaker在Nature上报道了他们的第八种核开关——一种能够催化自身回馈环路的核酶。
核开关（riboswitch）最初由耶鲁大学的分子生物学家Ronald Breaker和他的同事提出。在2004年10月8日的Science上，他们报道，最近他们又在细菌中发现了一种新的核开关，这种核开关能够开启一个甘油酸加工系统中三种蛋白的遗传调节。
Breaker在设计并合成一些对应于不同靶标化合物的“RNA开关”后，就开始着手寻找自然发生的核开关。新近在细菌中发现的这个核开关是他们到目前为止发现的第九种核开关。与其它的核开关不同，这种新的核开关类型是唯一能够与它的靶标发生“协同结合”的核开关。在这之前“协调结合”过程只常见于蛋白酶中。
这个发现令人惊异的地方还在于这种复杂的“RNA世界”残留还能在现代的生物体中看到。Breaker的发现也进一步说明RNA小分子对生命的复杂代谢的重要意义。
Fig. 1. Type I and type II gcvT motifs are natural RNA aptamers for glycine. (A) Consensus nucleotides present in more than 80% (black) and 95% (red) of representative sequences were identified by bioinformatics (17) (fig. S1). Circles and thick lines represent nucleotides whose base identities are not conserved. P1 through P4 identify common base-paired elements. ORF, open reading frame. (B) Patterns of spontaneous cleavage that occur with VC I-II in the absence and presence of glycine are depicted. Numbers adjacent to sites of changing spontaneous cleavage correspond to gel bands denoted with asterisks in (C) and data sets in (D). (C) Spontaneous cleavage products of VC I-II upon separation by polyacrylamide gel electrophoresis (PAGE) (7, 8) (fig. S2). NR, T1, and –OH represent no reaction, partial digest with RNase T1, and partial digest with alkali, respectively. Pre, precursor RNA. Some fragment bands corresponding to T1 digestion (cleaves after G residues) are labeled. Numbered asterisks identify locations of major structural modulation in response to glycine. The two rightmost lanes carry 1 mM of the amino acids noted. Brackets labeled I and II identify RNA fragments that correspond to cleavage events in the type I and type II aptamers, respectively. (D) Plots of the extent of spontaneous cleavage products versus increasing concentrations of glycine for aptamer I (sites 1 through 3), aptamer II (sites 5 through 7), and the linker sequence (site 4). C, concentration.
Fig. 2. Ligand specificity of VC II and VC I-II RNAs. (A) Inline probing of VC I-II in the absence (–) or presence of glycine (compound 1) or the analogs L-alanine (2), D-alanine (3), L-serine (4), L-threonine (5), sarcosine (6), mercaptoacetic acid (7), ß-alanine (8), glycine methyl ester (9), glycine tert-butyl ester (10), glycine hydroxamate (11), glycinamide (12), aminomethane sulfonic acid (13), and glycyl-glycine (14). Other notations are as described in the legend to Fig. 1C. (B) Equilibrium dialysis data for VC II and VC I-II (100 µM) in the absence (–) or presence (+) of excess (1 mM) unlabeled glycine, alanine, or serine as indicated. Fraction of 3H-glycine in chamber b reflects the amount of glycine bound by RNA plus half the total amount of free glycine in chambers a and b versus the total amount of 3H-glycine. i to iii, separate experiments where RNA and 3H are equilibrated (left) and competitor is subsequently added.
Fig. 3. Cooperative binding of two glycine molecules by the VC I-II RNA. Plot depicts the fraction of VC II (open) and VC I-II (solid) bound to ligand versus the concentration of glycine. The constant, n, is the Hill coefficient for the lines as indicated that best fit the aggregate data from four different regions (fig. S3). Shaded boxes demark the dynamic range (DR) of glycine concentrations needed by the RNAs to progress from 10%- to 90%-bound states.
Fig. 4. Control of B. subtilis gcvT RNA expression in vitro and in vivo. (A) The IGR between the yqhH and gcvT genes of B. subtilis encompassing both aptamers I and II was used for in vitro transcription and in vivo expression assays. Inline probing results were mapped, and mutations used to assess riboswitch function are indicated with red boxes. Orange shading identifies the putative intrinsic terminator stem, which is expected to exhibit mutually exclusive formation of aptamer II when bound to glycine. nt, nucleotide. (B) Single-round in vitro transcription assays demonstrating that full-length (Full) transcripts are favored when >10 µM glycine is added to the transcription mixture, whereas serine and most glycine analogs (fig. S5) are rejected by the riboswitch. The line reflects a best-fit curve to an equation reflecting cooperative binding with a Hill coefficient of 1.4 (19). An additional transcription product, termed "+," appears to be due to spurious transcription initiation (17). (C) Plot of the expression of a ß-galactosidase reporter gene fused to wild-type (WT) gcvT IGR or to a series of mutant IGRs (M1–M6). Data reflect the averages of three assays with two replicates each. Error bars indicate ± two standard deviations.
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