從Stanley Miller的放電實驗到“RNA世界”的提出，生命起源研究越來越受到科學界的重視,。近20年來在Science和Nature上發(fā)表的相關(guān)文章數(shù)以百計,，而Miller的放電實驗到RNA世界等生命起源的概念在學術(shù)界已經(jīng)眾所周知了。今年年初,，常年研究生命起源的Breaker在Nature上報道了他們的第八種核開關(guān)——一種能夠催化自身回饋環(huán)路的核酶,。
核開關(guān)（riboswitch）最初由耶魯大學的分子生物學家Ronald Breaker和他的同事提出。在2004年10月8日的Science上,，他們報道,，最近他們又在細菌中發(fā)現(xiàn)了一種新的核開關(guān),，這種核開關(guān)能夠開啟一個甘油酸加工系統(tǒng)中三種蛋白的遺傳調(diào)節(jié)。
Breaker在設(shè)計并合成一些對應于不同靶標化合物的“RNA開關(guān)”后,，就開始著手尋找自然發(fā)生的核開關(guān),。新近在細菌中發(fā)現(xiàn)的這個核開關(guān)是他們到目前為止發(fā)現(xiàn)的第九種核開關(guān)。與其它的核開關(guān)不同,，這種新的核開關(guān)類型是唯一能夠與它的靶標發(fā)生“協(xié)同結(jié)合”的核開關(guān),。在這之前“協(xié)調(diào)結(jié)合”過程只常見于蛋白酶中。
這個發(fā)現(xiàn)令人驚異的地方還在于這種復雜的“RNA世界”殘留還能在現(xiàn)代的生物體中看到,。Breaker的發(fā)現(xiàn)也進一步說明RNA小分子對生命的復雜代謝的重要意義,。
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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