Signal recognition particle receptor exposes the ribosomal translocon binding site

Signal sequences of secretory and membrane proteins are recognized by the signal recognition particle (SRP) as they emerge from the ribosome. This results in their targeting to the membrane by docking with the SRP receptor, which facilitates transfer of the ribosome to the translocon. Here, we present the 8 angstrom cryo-electron microscopy structure of a "docking complex" consisting of a SRP-bound 80S ribosome and the SRP receptor. Interaction of the SRP receptor with both SRP and the ribosome rearranged the S domain of SRP such that a ribosomal binding site for the translocon, the L23e/L35 site, became exposed, whereas Alu domain-mediated elongation arrest persisted.     

The crystal structure of the signal recognition particle in complex with its receptor

Cotranslational targeting of membrane and secretory proteins is mediated by the universally conserved signal recognition particle (SRP). Together with its receptor (SR), SRP mediates the guanine triphosphate (GTP)-dependent delivery of translating ribosomes bearing signal sequences to translocons on the target membrane. Here, we present the crystal structure of the SRP:SR complex at 3.9 angstrom resolution and biochemical data revealing that the activated SRP:SR guanine triphosphatase (GTPase) complex binds the distal end of the SRP hairpin RNA where GTP hydrolysis is stimulated. Combined with previous findings, these results suggest that the SRP:SR GTPase complex initially assembles at the tetraloop end of the SRP RNA and then relocalizes to the opposite end of the RNA. This rearrangement provides a mechanism for coupling GTP hydrolysis to the handover of cargo to the translocon.     

Structural basis for specific substrate recognition by the chloroplast signal recognition particle protein cpSRP43

Secretory and membrane proteins carry amino-terminal signal sequences that, in cotranslational targeting, are recognized by the signal recognition particle protein SRP54 without sequence specificity. The most abundant membrane proteins on Earth are the light-harvesting chlorophyll a/b binding proteins (LHCPs). They are synthesized in the cytoplasm, imported into the chloroplast, and posttranslationally targeted to the thylakoid membrane by cpSRP, a heterodimer formed by cpSRP54 and cpSRP43. We present the 1.5 angstrom crystal structure of cpSRP43 characterized by a unique arrangement of chromodomains and ankyrin repeats. The overall shape and charge distribution of cpSRP43 resembles the SRP RNA, which is absent in chloroplasts. The complex with the internal signal sequence of LHCPs reveals that cpSRP43 specifically recognizes a DPLG peptide motif. We describe how cpSPR43 adapts the universally conserved SRP system to posttranslational targeting and insertion of the LHCP family of membrane proteins.     

Crystal structure of an early protein-RNA assembly complex of the signal recognition particle

The signal recognition particle (SRP) is a universally conserved ribonucleoprotein complex that mediates the cotranslational targeting of secretory and membrane proteins to cellular membranes. A crucial early step in SRP assembly in archaea and eukarya is the binding of protein SRP19 to specific sites on SRP RNA. Here we report the 1.8 angstrom resolution crystal structure of human SRP19 in complex with its primary binding site on helix 6 of SRP RNA, which consists of a stem-loop structure closed by an unusual GGAG tetraloop. Protein-RNA interactions are mediated by the specific recognition of a widened major groove and the tetraloop without any direct protein-base contacts and include a complex network of highly ordered water molecules. A model of the assembly of the SRP core comprising SRP19, SRP54, and SRP RNA based on crystallographic and biochemical data is proposed.     

YGL9, encoding the putative chloroplast signal recognition particle 43 kDa protein in rice,is involved in chloroplast development

The nuclear-encoded light-harvesting chlorophyll a/b-binding proteins(LHCPs) are specifically translocated from the stroma into the thylakoid membrane through the chloroplast signal recognition particle(cp SRP) pathway. The cp SRP is composed of a cp SRP43 protein and a cp SRP54 protein, and it forms a soluble transit complex with LHCP in the chloroplast stroma. Here, we identified the YGL9 gene that is predicted to encode the probable rice cp SRP43 protein from a rice yellow-green leaf mutant. A phylogenetic tree showed that an important conserved protein family, cp SRP43, is present in almost all green photosynthetic organisms such as higher plants and green algae. Sequence analysis showed that YGL9 comprises a chloroplast transit peptide, three chromodomains and four ankyrin repeats, and the chromodomains and ankyrin repeats are probably involved in protein-protein interactions. Subcellular localization showed that YGL9 is localized in the chloroplast. Expression pattern analysis indicated that YGL9 is mainly expressed in green leaf sheaths and leaves. Quantitative real-time PCR analysis showed that the expression levels of genes associated with pigment metabolism, chloroplast development and photosynthesis were distinctly affected in the ygl9 mutant. These results indicated that YGL9 is possibly involved in pigment metabolism, chloroplast development and photosynthesis in rice.     

Crystal structure of the ribonucleoprotein core of the signal recognition particle

The signal recognition particle (SRP), a protein-RNA complex conserved in all three kingdoms of life, recognizes and transports specific proteins to cellular membranes for insertion or secretion. We describe here the 1.8 angstrom crystal structure of the universal core of the SRP, revealing protein recognition of a distorted RNA minor groove. Nucleotide analog interference mapping demonstrates the biological importance of observed interactions, and genetic results show that this core is functional in vivo. The structure explains why the conserved residues in the protein and RNA are required for SRP assembly and defines a signal sequence recognition surface composed of both protein and RNA.     

Requirement of GTP hydrolysis for dissociation of the signal recognition particle from its receptor.

The signal recognition particle (SRP) directs signal sequence specific targeting of ribosomes to the rough endoplasmic reticulum. Displacement of the SRP from the signal sequence of a nascent polypeptide is a guanosine triphosphate (GTP)-dependent reaction mediated by the membrane-bound SRP receptor. A nonhydrolyzable GTP analog can replace GTP in the signal sequence displacement reaction, but the SRP then fails to dissociate from the membrane. Complexes of the SRP with its receptor containing the nonhydrolyzable analog are incompetent for subsequent rounds of protein translocation. Thus, vectorial targeting of ribosomes to the endoplasmic reticulum is controlled by a GTP hydrolysis cycle that regulates the affinity between the SRP, signal sequences, and the SRP receptor.     

The structural basis of ribosome activity in peptide bond synthesis

Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs, we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site. Both substrate analogs are contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism of peptide bond synthesis appears to resemble the reverse of the acylation step in serine proteases, with the base of A2486 (A2451 in Escherichia coli) playing the same general base role as histidine-57 in chymotrypsin. The unusual pK(a) (where K(a) is the acid dissociation constant) required for A2486 to perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a buried phosphate that could stabilize unusual tautomers of these two bases. The polypeptide exit tunnel is largely formed by RNA but has significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e.     

Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor

The mechanism by which a signal recognition particle (SRP) and its receptor mediate protein targeting to the endoplasmic reticulum or to the bacterial plasma membrane is evolutionarily conserved. In Escherichia coli, this reaction is mediated by the Ffh/4.5S RNA ribonucleoprotein complex (Ffh/4.5S RNP; the SRP) and the FtsY protein (the SRP receptor). We have quantified the effects of 4.5S RNA on Ffh-FtsY complex formation by monitoring changes in tryptophan fluorescence. Surprisingly, 4.5S RNA facilitates both assembly and disassembly of the Ffh-FtsY complex to a similar extent. These results provide an example of an RNA molecule facilitating protein-protein interactions in a catalytic fashion.     

Insights into translational termination from the structure of RF2 bound to the ribosome

The termination of protein synthesis occurs through the specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site transfer RNA. Here we present, at a resolution of 3.5 angstroms, the crystal structure of RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon; it also suggests a model for the role of a universally conserved GGQ motif in the catalysis of peptide release.     

Heterodimeric GTPase core of the SRP targeting complex

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.     

灭幼脲和氟啶脲对斜纹夜蛾的毒力及其增效作用研究

在室内条件下测定了灭幼脲和氟啶脲对斜纹夜蛾(Prodenia litura Fabr)幼虫的毒力及其与5种增效剂的增效作用。结果发现,在3龄幼虫中,酯酶抑制剂TBPT对灭幼脲增效显著,SR值为8.58,羧酸酯酶抑制剂TPP增效6.04倍,谷胱甘肽转移酶抑制剂DEM增效4.22倍。多功能氧化酶抑制剂PB增效2.23倍,DDT脱氯化氢酶抑制剂DMC的增效作用最小,SR值为1.14,而在6龄幼虫中,以DEM对灭幼脲的增效作用最显著,SR值为6.4,其次为TBPT(5.93)>TPP(3.47)>DMC(1.49)。说明斜纺夜蛾对灭幼脲的解毒途径主要是水解作用,其次是氧化作用。就氟啶脲而言,5种增效剂无论是对3龄幼虫还是6龄幼虫,其增效比(SR)值和增效差(SD)值均无显著差异。     

Optimization of sowing date and seeding rate for high winter wheat yield based on pre-winter plant development and soil water usage in the Loess Plateau,China

Sowing date and seeding rate are critical for productivity of winter wheat(Triticum aestivum L.).A three-year field experiment was conducted with three sowing dates(20 September(SD1),1 October(SD2),and 10 October(SD3)) and three seeding rates(SR67.5,SR90,and SR112.5) to determine suitable sowing date and seeding rate for high wheat yield.A large seasonal variation in accumulated temperature from sowing to winter dormancy was observed among three growing seasons.Suitable sowing dates for strong seedlings before winter varied with the seasons,that was SD2 in 2012–2013,SD3 in 2013–2014,and SD2 as well as SD1 in 2014–2015.Seasonal variation in precipitation during summer fallow also had substantial effects on soil water storage,and consequently influenced grain yield through soil water consumption from winter dormancy to maturity stages.Lower consumption of soil water from winter dormancy to booting stages could make more water available for productive growth from anthesis to maturity stages,leading to higher grain yield.SD2 combined with SR90 had the lowest soil water consumption from winter dormancy to booting stages in 2012–2013 and 2014–2015; while in 2013–2014,it was close to that with SR67.5 or SR112.5.For productive growth from anthesis to maturity stages,SD2 with SR90 had the highest soil water consumption in all three seasons.The highest water consumption in the productive growth period resulted in the best grain yield in both low and high rainfall years.Ear number largely contributed to the seasonal variation in grain yield,while grain number per ear and 1 000-grain weight also contributed to grain yield,especially when soil water storage was high.Our results indicate that sowing date and seeding rate affect grain yield through seedling development before winter and also affect soil water consumption in different growth periods.By selecting the suitable sowing date(1 October) in combination with the proper seeding rate of 90 kg ha–1,the best yield was achieved.Based on these results,we recommend that the current sowing date be delayed from 22 or 23 September to 1 October.     

The mechanism for activation of GTP hydrolysis on the ribosome

Protein synthesis requires several guanosine triphosphatase (GTPase) factors, including elongation factor Tu (EF-Tu), which delivers aminoacyl-transfer RNAs (tRNAs) to the ribosome. To understand how the ribosome triggers GTP hydrolysis in translational GTPases, we have determined the crystal structure of EF-Tu and aminoacyl-tRNA bound to the ribosome with a GTP analog, to 3.2 angstrom resolution. EF-Tu is in its active conformation, the switch I loop is ordered, and the catalytic histidine is coordinating the nucleophilic water in position for inline attack on the γ-phosphate of GTP. This activated conformation is due to a critical and conserved interaction of the histidine with A2662 of the sarcin-ricin loop of the 23S ribosomal RNA. The structure suggests a universal mechanism for GTPase activation and hydrolysis in translational GTPases on the ribosome.     

贵州白香猪γ干扰素基因的克隆与序列分析

为获得贵州白香猪γ干扰素基因,以提取的经刀豆蛋白A诱导培养的贵州白香猪外周血淋巴细胞总RNA为模板,扩增全长猪γ干扰素基因,并克隆至pMD18-T Simple载体后测序分析。结果表明,pIFN-γ基因全长501bp,可编码166个氨基酸,其中前23位氨基酸为信号肽序列;pIFN-γ含有2个潜在N-糖基化位点;贵州白香猪与国内地方猪种之间pIFN-γ基因差异不大,具有很近的亲缘关系,其中与藏猪、成华猪、枫径猪、梅山猪的同源性达100%,而与内江猪的亲缘关系相对较远,与荣昌猪(DQ902588)亲缘最远。     

Many random sequences functionally replace the secretion signal sequence of yeast invertase

In the process of protein secretion, amino-terminal signal sequences are key recognition elements; however, the relation between the primary sequence of an amino-terminal peptide and its ability to function as an export signal remains obscure. The limits of variation permitted for functional signal sequences were determined by replacement of the normal signal sequence of Saccharomyces cerevisiae invertase with essentially random peptide sequences. Since about one-fifth of these sequences can function as an export signal the specificity with which signal sequences are recognized must be very low.     

磁性铁锆改性沸石覆盖对河道底泥磷释放的控制效果

通过批量吸附实验考察磁性铁锆改性沸石对水中磷酸盐的去除性能,再通过底泥培养实验考察磁性铁锆改性沸石覆盖对河道底泥磷释放的控制效果。结果表明:磁性铁锆改性沸石对水中磷酸盐的吸附能力良好。无论是在自然状态下,还是在缺氧控制状态下,河道底泥均向上覆水体中释放出一定数量的溶解性活性磷(SRP),而磁性铁锆改性沸石覆盖可以有效控制河道底泥中磷的释放,使得上覆水体中SRP浓度处于非常低的水平(0. 007～0. 031 mg/L)。当覆盖层受到破坏并使覆盖材料与表层底泥混合之后,磁性铁锆改性沸石仍然可以极大地降低上覆水体中SRP浓度,并且使底泥中潜在可移动态磷向较为稳定和非常稳定的磷形态转变,以及促使底泥中生物可利用性磷向非生物可利用性磷转变,使得底泥中磷发生释放的风险降低。以上结果初步表明,磁性铁锆改性沸石是一种非常有希望用于控制河道底泥中磷释放的覆盖材料。     

盐胁迫对芦苇愈伤组织抗氧化酶活性及脯氨酸含量的影响

[目的]探讨芦苇愈伤组织对盐胁迫的生理响应。[方法]以2种芦苇沙芦和水芦愈伤组织为试材,将其置于添加不同浓度NaCl的培养基进行胁迫处理,13 d后测定各项生理指标。[结果]50 mmol/L NaCl胁迫下水芦愈伤组织的细胞活力、TBARS以及活性氧含量均明显增加,而沙芦愈伤组织的则比对照略有降低;300 mmol/L NaCl胁迫下2种芦苇愈伤组织中TBARS与活性氧含量均有所增加;50和300mmol/L NaCl处理下水芦愈伤组织内脯氨酸含量增加,而沙芦愈伤组织的虽在50 mmol/L NaCl处理下降低,但在300 mmol/L NaCl处理下则远远高于水芦愈伤组织的;300 mmol/L NaCl处理下沙芦愈伤组织的4种抗氧化酶活性均显著升高,而水芦愈伤组织的SOD和POD活性虽有所增加,但增幅不如沙芦愈伤组织的。[结论]沙芦愈伤组织具有更强的耐盐性。