Báo cáo khoa học: Unusual metal specificity and structure of the group I ribozyme fromChlamydomonas reinhardtii23S rRNA

Unusual Metal Specificity and Structure of the Group I Ribozyme from Chlamydomonas reinhardtii 23S rRNA

Tác giả: Tai-Chih Kuo, Obed W. Odom, David L. Herrin

Lĩnh vực: Biochemistry, Molecular Cell and Developmental Biology

Nội dung tài liệu: Nghiên cứu này khám phá đặc tính kim loại bất thường và cấu trúc của ribozyme nhóm I, cụ thể là intron từ rRNA 23S LSU của Chlamydomonas reinhardtii (Cr.LSU). Phân tích động học cho thấy Mn²⁺ không ảnh hưởng đến sự gắn kết guanosine hoặc xúc tác, mà thay vào đó gây ra sự gấp sai của ribozyme một cách hợp lực cao. Cấu trúc bậc ba của intron được xem xét bằng phương pháp phân cắt Fe²⁺-EDTA, cho thấy một lõi ribozyme mở bất thường. Các phát hiện này có ý nghĩa đối với các đặc tính trong ống nghiệm và trong cơ thể sống của ribozyme intron này.

Mục lục chi tiết:

• Unusual metal specificity and structure of the group I ribozyme from Chlamydomonas reinhardtii 23S rRNA
• Keywords
• Correspondence
• Abbreviations
• Group I intron ribozymes require cations for folding and catalysis, and the current literature indicates that a number of cations can promote folding, but only Mg²⁺ and Mn²⁺ support both processes.
• However, some group I introns are active only with Mg²⁺, e.g. three of the five group I introns in Chlamydomonas reinhardtii.
• We have investigated one of these ribozymes, an intron from the 23S LSU rRNA gene of Chlamydomonas reinhardtii (Cr.LSU), by determining if the inhibition by Mn²⁺ involves catalysis, folding, or both.
• Kinetic analysis of guanosine-dependent cleavage by a Cr.LSU ribozyme, 23S.5AG, that lacks the 3′ exon and intron-terminal G shows that Mn²⁺ does not affect guanosine binding or catalysis, but instead promotes misfolding of the ribozyme.
• Surprisingly, ribozyme misfolding induced by Mn²⁺ is highly cooperative, with a Hill coefficient larger than that of native folding induced by Mg²⁺.
• At lower Mn²⁺ concentrations, metal inhibition is largely alleviated by the guanosine cosubstrate (GMP).
• The concentration dependence of guanosine cosubstrate-induced folding suggests that it functions by interacting with the G binding site, perhaps by displacing an inhibitory Mn²⁺.
• Because of these and other properties of Cr.LSU, the tertiary structure of the intron from 23S.5ΔGb was examined using Fe²⁺-EDTA cleavage.
• The ground-state structure shows evidence of an unusually open ribozyme core: the catalytic P3-P7 domain and the nucleotides that connect it to the P4-P5-P6 domain are exposed to solvent.
• The implications of this structure for the in vitro and in vivo properties of this intron ribozyme are discussed.
• Group I introns are cis-acting ribozymes whose substrates (5′ and 3′ splice sites) are attached intramolecularly.
• These introns have conserved uridine and guanosine nucleotides at the ends of the 5′ exon and intron segments, respectively.
• Although sequence conservation of group I introns is poor, their folded forms share a common core structure composed of two stacked-helix domains (P5–P4-P6 and P7-P3-P8) [1,2].
• Group I introns can be differentiated into five major subgroups (IA, IB, IC, ID, and IE) with further subdivisions that depend on the presence of peripheral domains that stabilize the core [3,4].
• Studies of several group I ribozymes, but especially the intron from the large rRNA gene of Tetrahymena thermophila (Tt.LSU), indicate that some domains are modular, and that the catalytic site is buried inside the folded ribozyme [5-7].
• The tertiary structure is stabilized by domain-domain interactions, such as hydrogen bonding of loop-receptor pairs, base triples, and pseudoknots [1,2].
• The group I self-splicing pathway consists of two consecutive transesterification reactions with the activated phosphodiesters at the splice sites.
• First, the 3′-OH of an exogenous guanosine nucleotide (GTP)
• Mn2+ inhibition and structure of Cr.LSU ribozyme
• Results
• Inhibition of self-splicing and G-dependent cleavage by Mn2+
• In splicing reactions with 23S.5 pre-RNA, substituting part (> 1/3) of the Mg2+ with Mn2+ reduced the amount of products, which were undetectable when Mn2+ was the only divalent cation (not shown [26]).
• Varying the Mn2+ concentration (0.1–50 mm), pH (5.5-7.5), monovalent salt, temperature (37 or 47 °C), and reaction time (0.25-60 min) also did not yield any splicing products (data not shown).
• Mn2+ inhibits self-splicing of the 23S.3 and 23S.4 pre-RNAs, which have different lengths of 5′ exon [23], and it inhibits a trans-reaction [10] that involves the free intron reacting with 5.8S rRNA (not shown).
• Together, these data suggested that inhibition by Mn2+ probably involved the core ribozyme, and not the intron open reading frame (ORF) or exon sequences.
• 23S.5ΔGb pre-RNA is a truncated version of 23S.5 that terminates 3 nucleotides before the end of the intron (Fig. 1A; see Fig. 5D for the intron sequence and structure).
• Core catalytic activity is preserved, however, in the form of G-dependent cleavage at the 5′ splice site.
• Incubation of 23S.5AG with saturating Mg2+ (25 mm) and GMP (150 µm) produces the InAG and 5′ exon (not shown) molecules as expected (Fig. 1B).
• In the presence of 10 mm Mn2+, however, less of the pre-RNA reacts (compare Fig. 1B,C).
• Thus, the inhibition of 23S.5 self-splicing by Mn2+ is recapitulated by the G-dependent cleavage of 23S.5AGb pre-RNA.
• Mixed-metal titrations over a range of total metal concentrations showed that the ratio of the two metals is somewhat more important than the absolute concentrations; a ratio of Mn2+/Mg2+ of approximately 1:2 or higher inhibited G-dependent cleavage of 23S.5AG RNA (and self-splicing of 23S.5, not shown).
• Quantification of time-course reactions similar to those in Fig. 1B,C, except at two different GMP concentrations (Fig. 1D), show that approximately 85% of the 23S.5AG♭ RNA is kinetically homogeneous and highly active (kobs approximately 0.9 min¯¹ at 150 μμ GMP).
• The remaining fraction (approximately 15%) is relatively inactive, reacting 20-30 times more slowly (kobs = 0.032 min¯¹ at 150 μμ GMP, and 0.017 min¯¹ at 20 µm GMP).
• It can also be inferred from Fig. 1D that the inactive RNA fraction increases substantially when 10 mm Mn2+ is added, from 15% to 32% at 150 μμ GMP, or 50% at 20 µm GMP.
• The inverse is true for the active fraction, which decreased from 85% to 68% and 50%, respectively.
• The observed rate of G-dependent cleavage by the active fraction is not substantially affected by Mn2+: the kobs at 150 µm GMP is 0.96 min¯¹ with Mn2+ and 0.87 min¯¹ without it, and at 20 µm GMP, the kobs is 0.49 min¯¹ with Mn2+ and 0.55 min¯¹ without it.
• We conclude that Mn2+ increases the proportion of 23S.5ΔG♭ pre-RNA that is inactive, whereas GMP increases the proportion that is active.
• An extensive kinetic analysis was performed at 5–300 μμ GMP and 10 or 15 mm Mn2+ in the presence of 25 mm Mg2+.
• Figure 2A shows that Mn2+ has little or no effect on the K&1/2 or keat for the active fraction of the ribozyme.
• However, as Fig. 2B shows quite dramatically, the metal decreases the size of this fraction.
• It should be noted that the proportion of active ribozyme without Mn2+ is approximately 88% at all GMP concentrations tested.
• In the presence of 10 mm Mn2+, the maximum size of this fraction is 66% (> 100 μμ GMP), and it decreases dramatically at GMP concentrations < 100 μμ (Fig. 2B).
• At 15 mm Mn2+, the percentage of active ribozyme is even lower (approximately 30% at > 25 µm GMP) and it decreases further at GMP < 20 μm.
• These data extend the above result, and support the conclusion that Mn2+ affects mainly the correct folding of the ribozyme.
• The results also show that most of the inhibition by 10 mм Mn2+ is reversed by saturating GMP (Fig. 2B).
• Moreover, the fact that this GMP activation curve is similar to the GMP cleavage plot (Fig. 2A) indicates that GMP is promoting ribozyme folding via the G binding site in P7.
• The rate of G-dependent cleavage by the inactive fraction that forms in Mg2+ increases slowly with GMP concentration (K1/2 = 60 µm and kcat = 0.035 min¯¹, not shown).
• However, the inactive fraction in Mn2+ (10 mm in Fig. 2B) reacts much more slowly (approximately 0.007 min¯¹) and independently of the GMP concentration (not shown).
• This result suggests that Mn2+ induces a distinctive slow-reacting fraction that must go through a rate-limiting conformational change before it can bind GMP and catalyze cleavage.
• Model for the effect of Mn2+ on the 23S.5 Gb ribozyme
• Since Mn2+ does not substantially affect the kinetic parameters for the active ribozyme, but instead reduces the size of this fraction, the following scheme (Scheme 1) is proposed to describe the inhibition of G-dependent cleavage by the metal ion.
• U + nMg2+ ≒ pre-RNA-nMgative
• U + mMn2+ = pre-RNA.mMninactive
• Scheme 1
• U is unfolded 23S.5AGb pre-RNA, and the binding of a minimum of n Mg2+ ions leads to formation of the active complex, whereas binding of a minimum of m Mn2+ ions forms the inactive complex.
• The sizes of the active and inactive fractions are the result of competitive metal binding to RNA.
• The values of n and m are estimated from Hill analysis of G-dependent cleavage of 23S.5AG.
• It should be noted that Scheme 1 indicates only the initial and final states of the pre-RNA; it does not invoke or rule out any misfolded intermediates that might form.
• To determine n, G-dependent cleavage of 23S.5AGb was analyzed at varying MgCl2 concentrations (0-50 mm) and either 0 or 12 mm MnCl2 (plus saturating GMP).
• Figure 3A shows that in the presence of Mn2+ a much higher concentration of Mg2+ is required to form the same amount of cleavage product (InAG).
• A quantitative analysis of similar experiments (Fig. 3B), but using 0–100 mm Mg2+ and several fixed Mn2+ concentrations (0, 7, 12 and 17 mm), reveals that cleavage increases cooperatively with increasing Mg2+, in the absence or presence of Mn2+.
• The midpoint of the Mg2+ titration curve in the absence of Mn2+ is approximately 4.5 mm, and nearly full activity is reached by 10 mm Mg2+.
• Hill analysis of the data gives n-values of 2.6, 2.2, 2.7 and 2.7, for reactions in 0, 7, 12 and 17 mm Mn2+ respectively.
• These results indicate that formation of the active RNA-Mg2+ complex involves the binding of at least three Mg2+ ions by the ribozyme.
• The data also show that Mg2+ can completely block the inhibition caused by Mn2+.
• Fig. 2. Ribozyme activity at varying GMP and fixed Mn2+ concentrations.
• The G-dependent cleavage reactions were performed at different concentrations of GMP (0-300 µm) and Mn2+ (0, 10 or 15 mm) in the presence of 25 мм MgCl2.
• The reactions were analyzed as described in Experimental procedures, and the observed rate constants (A) and percentages (B) of active ribozyme were plotted versus GMP concentration.
• In (A), the line was fitted using the 0 mm Mn2+ data.
• In (B), the kcat values are 1.1, 1.0 and 1.0 min¯¹, respectively, for the reactions at 0, 10 and 15 мм Mn2+, and the corresponding K1/2 values are 22, 24 and 21 µm, respectively.
• Fig. 3. Mg2+ dependence of ribozyme activity at fixed Mn2+ concentrations.
• (A) G-dependent cleavage of 23S.5ΔGb pre-RNA at varying Mg2+ concentration, and 0 mm (top) or 12 mm (bottom) MnCl2; the reactions also contained 150 μμ GMP, and were incubated for 40 s.
• They were separated on a denaturing polyacrylamide gel, which was phosphorimaged.
• (B) Mg2+ concentration curves at fixed Mn2+ concentrations.
• G-dependent cleavage of 23S.5 Gb was performed as in (A), except for using the indicated Mn2+ concentrations.
• The cleavage product (InAGb) was quantified, and expressed as a percentage of total RNA [Relative In∆Gb (%)].
• The data were curve-fitted to obtain Hill coefficients as described in Experimental procedures.
• To determine the minimal number of Mn2+ involved in forming the inactive RNA-metal complex, G-dependent cleavage reactions were performed at varying Mn2+ and fixed Mg2+ concentrations.
• Figure 4A shows representative gels of reactions that were performed at varying (0-30 mm) MnCl2 con- centrations and either 15 or 25 mm MgCl2.
• Fig. 4. Mn2+ dependence of ribozyme inhibition at fixed Mg2+ concentrations.
• G-dependent cleavage of 23S.5 Gb pre-RNA was performed with the indicated concentrations of MnCl2 (0-30 мм), 10 or 25 mM MgCl2, and 150 μμ GMP for 40 s.
• The reactions were analyzed as in Fig. 3.
• GMP-dependent cleavage decreases sharply above 5 and 7 mm MnCl2, respectively.
• Quantitative analysis (Fig. 4B) gives a Hill value (m) of 5.7 for the experiments with 15 and 25 mM MgCl2.
• Thus, formation of the inactive ribozyme is highly cooperative.
• The data also suggest that binding of a minimum of six Mn2+ ions is involved in the misfolding that forms the inactive ribozyme.
• Structure of the Cr.LSU intron in the 23S.5 Gb pre-RNA
• The unusual metal specificity, as well as other atypical features of Cr.LSU (see Discussion), led us to study the global tertiary structure of the intron using hydroxyl radical cleavage.
• This analysis was carried out by incubating end-labeled, Mg2+-folded intron (InΔGb) with Fe2+-EDTA [7,8].
• It should be emphasized that the RNA structure revealed by Fe2+-EDTA is an averaged image of the RNA molecules in solution.
• However, since the kinetic data indicate that 85-90% of 23S.5AG♭ pre-RNA is functionally similar, we assume that the predominant signal in the protection pattern is from active ribozyme.
• Figure 5A shows an Fe2+-EDTA cleavage analysis performed at 0–25 mm Mg2+.