RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway

Ribonuclease mitochondrial RNA processing (RNase MRP) is a multifunctional ribonucleoprotein (RNP) complex that is involved in the maturation of various types of RNA including ribosomal RNA. RNase MRP consists of a potential catalytic RNA and several protein components, all of which are required for cell viability. We show here that the temperature-sensitive mutant of rmp1, the gene for a unique protein component of RNase MRP, accumulates the dimeric tRNA precursor, pre-tRNA^Ser-Met. To examine whether RNase MRP mediates tRNA maturation, we purified the RNase MRP holoenzyme from the fission yeast Schizosaccharomyces pombe and found that the enzyme directly and selectively cleaves pre-tRNA^Ser-Met, suggesting that RNase MRP participates in the maturation of specific tRNA in vivo. In addition, mass spectrometry–based ribonucleoproteomic analysis demonstrated that this RNase MRP consists of one RNA molecule and 11 protein components, including a previously unknown component Rpl701. Notably, limited nucleolysis of RNase MRP generated an active catalytic core consisting of partial mrp1 RNA fragments, which constitute “Domain 1” in the secondary structure of RNase MRP, and 8 proteins. Thus, the present study provides new insight into the structure and function of RNase MRP.     

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34^cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13^suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34^cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34^cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.     

Interaction with Cfd1 Increases the Kinetic Lability of FeS on the Nbp35 Scaffold

P-loop NTPases of the ApbC/Nbp35 family are involved in FeS protein maturation in nearly all organisms and are proposed to function as scaffolds for initial FeS cluster assembly. In yeast and animals, Cfd1 and Nbp35 are homologous P-loop NTPases that form a heterotetrameric complex essential for FeS protein maturation through the cytosolic FeS cluster assembly (CIA) pathway. Cfd1 is conserved in animals, fungi, and several archaeal species, but in many organisms, only Nbp35 is present, raising the question of the unique roles played by Cfd1 and Nbp35. To begin to investigate this issue, we examined Cfd1 and Nbp35 function in budding yeast. About half of each protein was detected in a heterocomplex in logarithmically growing yeast. Nbp35 readily bound ⁵⁵Fe when fed to cells, whereas ⁵⁵Fe binding by free Cfd1 could not be detected. Rapid ⁵⁵Fe binding to and release from Nbp35 was impaired by Cfd1 deficiency. A Cfd1 mutation that caused a defect in heterocomplex stability supported iron binding to Nbp35 but impaired iron release. Our results suggest a model in which Cfd1-Nbp35 interaction increases the lability of assembled FeS on the Nbp35 scaffold for transfer to target apo-FeS proteins.     

Regulation of maturation-promoting factor by protein kinase C in Chaetopterus oocytes

Summary

We present the results of a variety of studies showing that activation of protein kinase C (PKC) in oocytes of Chaetopterus pergamentaceus results in germinal vesicle breakdown (GVBD). Phorbol esters and diacylglycerol can initiate a morphologically normal GVBD accompanied by a spectrum of associated biochemical processes, including increased protein phosphorylation, a shift in protein synthesis and activation of a protein kinase, maturation promoting factor (MPF). MPF activation is essential for GVBD in response to phorbol esters. In addition, inhibitors of PKC can block naturally-induced GVBD. We also present evidence that PKC can phosphorylate p34^cde2, the catalytic subunit of MPF and that phosphorylation by PKC increases the histone H1 kinase activity of immunoprecipitated MPF. Immunoblot studies show that Chaetopterus oocyte p34^cdc2 is not tyrosine phosphorylated prior to the initiation of GVBD, indicating that activation of MPF at GVBD in this species does not require p80^cdc25, the activator of MPF at mitosis. These results suggest that PKC is an essential regulator of GVBD which can directly phosphorylate and regulate p34^cdc2. Since PKC is the intracellular receptor for and is directly activated by tumor-promoters, tumor promotion might involve acceleration of the cell cycle through modification of the enzymatic activity of MPF by PKC.     

Cuticle protein in adult Locusta migratoria

The total protein, chitin and soluble protein content of the abdominal cuticle of male adult locusts was analysed. After an initial period of increase, cuticular protein and chitin content levelled off with the onset of sexual maturation. After ecdysis, the amount of soluble cuticular protein increased unitil maturation. Specific cuticular protein electrophoretic bands decreased in staining intensity with development and were presumed to become bound within the cuticle. Incorporation of ³H-leucine into cuticular proteins was highest into pharate adult cuticle, then decreased after ecdysis to a constant level after maturation. Following sexual maturation when the total cuticular protein and soluble protein content remained constant incroporation of ³H-leucine continued indicating the dynamic nature of the cuticle.     

Influence of exogenous ions on the events of maturation in Rana pipiens oocytes

Full-grown ovarian oocytes removed from non-hormone-treated Rana pipiens females exhibit a low level of protein synthesis, the rate of which is dependent upon the ionic environment. The highest rates of protein synthesis in these oocytes are obtained in media containing either a divalent cation (Ca⁺⁺ or Mg⁺⁺) or high levels of K⁺. The dependence of protein synthesis on ionic environment persists through about the first 18-24 hours of maturation (at 18°C). Normal maturation of oocytes in vitro also has specific ionic requirements for the first 24 hours. In this case, the process requires high ionic strength (T/2 = 1.0-1.2) and divalent cations. The kinetics of K⁺ exchange suggest that K⁺ exists in the ovarian oocyte in two compartments; one in equilibrium with the exogenous medium and freely exchangeable; the other in equilibrium with the exogenous medium and freely exchangeable; the other in equilibrium with the first internal compartment and only very slowly exchangeable. The slowly exchangeable (bound) compartment contains about 95% of all endogenous K⁺. In hormone stimulated oocytes, the kinetics of K⁺ exchange are essentially the same. Oocyte adaptation to ionic environment is discussed as a possible regulatory mechanism during maturation.     

Endogenous phosphorylated proteins during maturation of Xenopus laevis oocytes

During the course of maturation of Xenopus laevis oocyte a burst of phosphorylation occurs around germinal vesicle breakdown. At the same time a relative drop in a unique phosphoprotein (protein I; mot wt ～40,000) is observed. Enucleation of [³²P] labeled oocytes has shown the cytoplasmic localization of protein I. Methylxanthines and cholera toxin, which inhibit progesterone-induced maturation, block the burst of phosphorylation and do not change the amount or the distribution of [³²P] phosphoproteins.     

Cloning of Dictyostelium eIF6 (p27) and mapping its nucle(ol)ar localization subdomains

Eukaryotic translation initiation factor 6 (eIF6), also termed p27^BBP, is an evolutionary conserved regulator of ribosomal function. The protein is involved in maturation and/or export from the nucleus of the 60S ribosomal subunit. Regulated binding to and release from the 60S subunit also regulates formation of 80S ribosomes, and thus translation. The protein is also found in hemidesmosomes of epithelial cells expressing β4 integrin and is assumed to regulate cross-talk between β4 integrin, intermediate filaments and ribosomes. In the present study we show that the Dictyostelium eIF6 (also called p27^BBP) gene is expressed during growth, down-regulated during the first hours of starvation, and up-regulated again at the end of aggregation. Phagocytosis, and to a lesser extent pinocytic uptake of axenic medium, stimulate gene expression in starving cells. The eIF6 gene is present in single copy and its ablation is lethal. We utilized the green fluorescent protein (GFT) as fusion protein marker to investigate sequences responsible for eIF6 subcellular localization. The protein is found both in cytoplasm and nucleus, and is enriched in nucleoli. Deletion sequence analysis shows that nucle(ol)ar localization sequences are located within the N- and C-terminal subdomains of the protein.     

The Mycobacterium avium subsp. Paratuberculosis protein MAP1305 modulates dendritic cell-mediated T cell proliferation through Toll-like receptor-4

In this study, we show that Mycobacterium avium subsp. Paratuberculosis MAP1305 induces the maturation of bone marrow-derived dendritic cells (BMDCs), a representative antigen presenting cell (APC). MAP1305 protein induces DC maturation and the production of pro-inflammatory cytokines (Interleukin (IL)-6), tumor necrosis factor (TNF)-α, and IL-1β) through Toll like receptor-4 (TLR-4) signaling by directly binding with TLR4. MAP1305 activates the phosphorylation of MAPKs, such as ERK, p38MAPK, and JNK, which is essential for DC maturation. Furthermore, MAP1305-treated DCs transform naïve T cells to polarized CD4⁺ and CD8⁺ T cells, thus indicating a key role for this protein in the Th1 polarization of the resulting immune response. Taken together, M. avium subsp. Paratuberculosis MAP1305 is important for the regulation of innate immune response through DC-mediated proliferation of CD4⁺ and CD8⁺ T cells. [BMB Reports 2014; 47(2): 115-120]     

Epidermal Growth Factor-Mediated Mitogen-Activated Protein Kinase3/1 Pathway Is Conducive to In Vitro Maturation of Sheep Oocytes

Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo’s capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10⁻⁴ mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10⁻⁶ mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10⁻⁴ mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10⁻⁶ mol/l U0126 group and 42.6% in 10⁻⁴ mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10⁻⁴ mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes.     

The methodological approach for the generation of humandendritic cells from monocytes affects the maturation state of the resultant dendritic cells

Dendritic cells (DCs) are effective as antigen-presenting cells in the immune system and are present at two functional stages depending on their maturation state. For experimental investigation of this concept, CD14⁺ monocytes from blood are isolated and cultured to generate in vitro the DCs needed for functional analysis. For positive selection of CD14⁺ monocytes we compared two immunomagnetic bead technologies: MACS^® Separation, created by Miltenyi Biotec, and EasySep^® Selection, created by StemCell Technologies. The monocytes provided dendritic cells for their functional analysis. Lipopolysaccharide was added to cultured DCs to induce maturation. Although both systems generated DCs from the positively selected CD14⁺ cells, there were certain differences between them. Morphological, phenotypic, and functional analysis showed that MACS^®-selection provided DCs that have typical features corresponding to day 6 or 7 of maturation. EasySep^®–DCs exist in a partially-mature state from day 6 onward, even without the addition of a maturation stimulus. The reason behind this partial maturation is possibly based on the dextran-coated beads that are associated with the EasySep^® product. Both methods provide pure and viable DCs, but we would recommend using the MACS^® system for obtaining DCs suitable for functional studies.     

1H, 13C and 15N backbone and side-chain resonance assignments of reduced CcmG from Escherichia coli

CcmG is a periplasmic, membrane-anchored protein widely distributed in a variety of species. In Escherichia coli, the CcmG protein always acts as a weak reductant in the electron transport chain during cytochrome c maturation (Ccm). Here we report ¹H, ¹⁵N and ¹³C backbone and side-chain resonance assignments of the reduced CcmG protein (residues 19–185, renumbered as 1–167) from E. coli. This work lays the essential basis for the further structural and functional analysis of reduced CcmG.     

Program of early development in the mammal: changes in patterns and absolute rates of tubulin and total protein synthesis during oogenesis and early embryogenesis in the mouse.

The absolute rates of total protein synthesis and tubulin synthesis during oogenesis and early embryogenesis in the mouse have been determined by measuring specific activities of the endogenous methionine pool and rates of incorporation of [³⁵S]methionine into total protein and tubulin. The absolute rate of protein synthesis decreases from 43 to 33 pg/hr/oocyte during meiotic maturation, while the size of the endogenous methionine pool remains essentially unchanged at 65 fmole/oocyte (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978, Proc. Nat. Acad. Sci. USA,75, 4160). The one-cell mouse embryo synthesizes protein at a rate of 45 pg/hr/embryo, so that fertilization is accompanied by about a 40% increase in the absolute rate of total protein synthesis. The eight-cell compacted embryo synthesizes protein at the rate of 51 pg/hr/embryo. The size of the endogenous methionine pool increases dramatically during early embryogenesis, from 74 fmole in the unfertilized ovum to 137 and 222 fmole in the one-cell embryo and eight-cell compacted embryo, respectively. Tubulin is one of the major proteins synthesized by the mouse oocyte and embryo since the absolute rate of tubulin synthesis is, on the average, 1.3% that of total protein synthesis. The absolute rate of tubulin synthesis decreases from 0.61 to 0.36 pg/hr/oocyte during meiotic maturation and then increases to 0.60 pg/hr/embryo in the one-cell embryo and to 0.66 pg/hr/embryo in the eight-cell compacted embryo. During meiotic maturation and early embryogenesis the direction and magnitude of changes in the rate of tubulin synthesis closely parallel those of total protein synthesis. Although equimolar amounts of tubulin subunits are present in microtubules, the ratio of the absolute rate of synthesis of the β subunit to that of the α subunit is about 2.0 throughout meiotic maturation and early embryogenesis.High-resolution two-dimensional gel electrophoretic analysis of [³⁵S]methionine-labeled proteins reveals that many of the newly synthesized proteins that first appear during meiotic maturation of the oocyte continue to be synthesized in the one-cell embryo. Nearly all of the proteins synthesized in the one-cell embryo are also synthesized in the unfertilized ovum, although some changes in the pattern of protein synthesis are associated with fertilization. Therefore, the developmental program for early embryogenesis in the mouse appears to be activated during meiotic maturation of the oocyte. These results are compared with those obtained using oocytes and embryos from nonmammalian animal species.     

Effects of Mitochondrial Protein Synthesis Inhibitors on the Incorporation of Isoleucine into Plasmodium falciparum In Vitro1

Plasmodium falciparum was grown in human erythrocytes in vitro and the effect of chloramphenicol, erythromycin, and tetracycline on growth and maturation of the parasites and on their ability to incorporate [³H]isoleucine into protein was observed. Exposure of rings to high concentrations of chloramphenicol had little effect on subsequent maturation of the rings whereas brief (4 h) exposure of trophozoites caused a dose-dependent inhibition of subsequent ring formation. Incorporation of [³H]isoleucine into protein was not affected during at least 6 h of exposure to high concentration of the three drugs examined, but appreciable inhibition was observed after 21 h, with chloramphenicol being the least effective inhibitor. These results suggest that there is a stage-specific effect of inhibition of mitochondrial protein synthesis on subsequent development and that the mitochondria are essential for growth and development even though they lack a functional Krebs cycle.     

Role of calcium in the localization of maternal poly(A)+RNA and tubulin mRNA in Xenopus oocytes

Summary Poly(A)⁺RNA and tubulin mRNA are localized in the periphery of Xenopus oocytes and become delocalized during meiotic maturation. Delocalization of this RNA can be triggered by incubation in agents which reduce entry of calcium ions into the cell (e.g. lanthanum chloride and verapamil). Although these agents ordinarily promote meiotic maturation, addition of theophylline to the medium will inhibit maturation but not delocalization. Manipulations which prevent calcium entry without inducing meiotic maturation (e.g. calcium-free buffer) are also shown to trigger disruption of the RNA localization. In addition, manipulations which reduce chloride efflux from the cell (e.g. increasing the external chloride ion concentration with choline chloride) result in disruption of the localization of poly (A)⁺ RNA and tubulin mRNA without inducing meiotic maturation. The calcium-dependent chloride efflux present in Xenopus oocytes disappears after the oocyte has been stimulated to proceed through meiotic maturation. We show that reduction of the influx of calcium ions or efflux of chloride ions induces the delocalization of poly (A)⁺RNA and tubulin mRNA without inducing meiotic maturation. We suggest, therefore, that reducing the transmembrane movement of these ions is likely to be the natural trigger for the delocalization of poly(A)⁺RNA and tubulin mRNA.     

Effects of the addition of oocyte meiosis-inhibiting drugs on the expression of maturation-promoting factor components and organization of cytoplasmic organelles

The present study evaluated the effects of the blockade of meiosis in bovine oocytes by the cyclin-dependent kinase inhibitors roscovitine (ROS) and butyrolactone-I (BL-I) on nuclear maturation and extracellular signal-regulated kinase 1/2 (ERK1/2), cyclin B1 and p34^cdc2 protein expression and localization. We also evaluated ultrastructural changes in oocytes. Immature oocytes were obtained from slaughtered bovines and divided into: (1) control (oocytes for in vitro maturation only in tissue culture medium-199 for 24 h), (2) oocytes that were treated with 12.5μMROS for 6 h, (3) oocytes that were treated with 50μMBL-I for 6 h and (4) oocytes that were treated with 6.25 μMROS+25 μMBL-I for 6 h. Incubation with inhibitors was followed by the reversal of blockade for 18 h. Oocytes then underwent immunohistochemical analysis to visualize chromatin and assess ERK1/2, cyclin B1 and p34^cdc2 localization/expression, followed by preparation of the cells for ultrastructure analysis by electron microscopy. The groups at 6 h of maturation and before IVM exhibited the lowest number of oocytes in metaphase I. ROS group had the highest number of degenerating oocytes (p < 0.05). After maturation, majority of oocytes were in metaphaseII with no differences among groups (p> 0.05). ERK1/2, cyclin B1 and p34^cdc2 expression differed throughout inhibition and oocyte maturation (p < 0.05). No difference was observed in the localization of these proteins in the ooplasm. No ultrastructural changes in oocytes were observed between treatments, with the exception of treatment with drugs that augmented lipid metabolism (p < 0.05). Results indicate that the effects of CDK1 inhibitors are reversible in bovine oocytes, indicated by nuclear, cytoplasmic, and molecular maturation parameters.     

Structure of T4 polyheads: II. A pathway of polyhead transformations as a model for T4 capsid maturation

We have studied the aberrant tubular polyheads of bacteriophages T4D and T2L as a model system for capsid maturation. Six different types of polyhead surface lattice morphology, and the corresponding protein compositions are reported and discussed. Using in vitro systems to induce transformations between particular polyhead types, we have deduced that the structural classes represent successive points in a transitional pathway. In the first step, coarse polyheads (analogous to the prohead τ-particle) are proteolytically cleaved by a phagecoded protease, a fragment of the gene 21 product. This cleavage of P23 to P23¹ induces a co-operative lattice transformation in the protein of the surface shell, to a conformation equivalent to that of T2L giant phage capsids. These polyheads (derived either from T4 or T2L lysates) can accept further T4-coded proteins. In doing so, they pass through intermediate structural states, eventually reaching an end point whose unit cell morphology is indistinguishable from that of the giant T4 capsids. At least one protein (called soc (Ishii & Yanagida, 1975)) is bound stoichiometrically to P23¹ in the end-state conformation. The simulation of several aspects of capsid maturation (cleavage of P23 to P23¹, stabilization, and lattice expansion) in the polyhead pathway suggest that it parallels the major events of phage T-even capsid maturation, decoupled from any involvement of DNA packaging.     

Localization of Qβ Maturation Cistron Ribosome Binding Site

THE RNA of phage Qβ consists of about 3,500 nucleotides¹ and comprises three known cistrons coding for maturation protein (a structural component of the viral particle also designated as A or A₂ protein), coat protein and the β subunit of the viral replicase². When ribosomes are bound to intact Qβ RNA they attach predominantly to one binding site, namely that at the beginning of the coat cistron³. This binding site is located at about the 1,400th nucleotide from the 5′ end¹.     

Expression and localization of malate synthase during maturation and dessication of castor bean seeds

Summary Enzymatic levels and subcellular localization of malate synthase in maturing seeds of castor bean (Ricinus communis cv. Hale) are reported. Extracts of maturing seeds exhibited moderately high specific activity (9.68 nmoles/min/mg protein) at 15–20 DAP and lower specific activity (0.49) in mature, dry seeds. Subcellular localization of the enzyme during seed maturation was primarily cytosolic (85%). The remainder of the activity in sucrose gradients was located at high density (1.21 g/cm³). Dry seeds did not contain organelle-bound malate synthase activity. In extracts of 4-day germinated seeds the enzyme was present at high specific activity (12.8 nmoles/min/mg protein) with better than 85% of the total activity in glyoxysomes (1.24 g/cm³).Two polypeptides, 62kDa and 66kDa, reactive with anti-malate synthase were detected at high density in sucrose gradients of homogenates of late-maturing seeds (60 DAP); dry seeds; and seeds imbibed for 6 h. One polypeptide, 62 kDa, in 4-day germinated seeds, reacted with anti-malate synthase. Immunoreactive polypeptides in late-maturing and dry seeds were present at approximately 1/760 of the level found in 4-day germinated seeds. We conclude that malate synthase activity is prominent during early seed maturation but is very low and minimally compartmentalized during late maturation. The rapidly sedimenting immunoreactive polypeptides from dry seeds are enzymatically inactive and are presumed to be of no physiological significance.Abbreviations DAP days after pollination - MS malate synthase - EDTA ethylenediamine tetraacetic acid - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis - BSA bovine serum albumin - IgG gamma globulin     

Maturation Mechanism of Severe Acute Respiratory Syndrome (SARS) Coronavirus 3C-like Proteinase

The 3C-like proteinase (3CL^pro) of the severe acute respiratory syndrome (SARS) coronavirus plays a vital role in virus maturation and is proposed to be a key target for drug design against SARS. Various in vitro studies revealed that only the dimer of the matured 3CL^pro is active. However, as the internally encoded 3CL^pro gets matured from the replicase polyprotein by autolytic cleavage at both the N-terminal and the C-terminal flanking sites, it is unclear whether the polyprotein also needs to dimerize first for its autocleavage reaction. We constructed a large protein containing the cyan fluorescent protein (C), the N-terminal flanking substrate peptide of SARS 3CL^pro (XX), SARS 3CL^pro (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CL^pro using fluorescence resonance energy transfer. In contrast to the matured 3CL^pro, the polyprotein, as well as the one-step digested product, 3CLP-Y-His, were shown to be monomeric in gel filtration and analytic ultracentrifuge analysis. However, dimers can still be induced and detected when incubating these large proteins with a substrate analog compound in both chemical cross-linking experiments and analytic ultracentrifuge analysis. We also measured enzyme activity under different enzyme concentrations and found a clear tendency of substrate-induced dimer formation. Based on these discoveries, we conclude that substrate-induced dimerization is essential for the activity of SARS-3CL^pro in the polyprotein, and a modified model for the 3CL^pro maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.