Identiﬁcation and Characterization of Elf1, a Conserved Transcription Elongation Factor in Saccharomyces cerevisiae-猫眼课题宝

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为了确定以前未知的转录延长因子，进行了基因筛查，以确定当与编码延长因子TFIIS和Spt6的基因突变结合时导致致命性的突变。这张屏幕发现了YKL160W的一个突变，此后被命名为ELF1(ElongationﬁFor Actor 1)。进一步的分析证实了Elf1(Cid：1)突变和编码几个已知延长因子的基因突变之间的合成致死性，这些基因包括Spt4、Spt5、Spt6和Paf1复合体的成员。全基因组合成致死性研究表明，Elf1(Cid：1)ﬁ特异地与影响转录延长的基因突变相互作用。染色质免疫沉淀实验表明，Elf1是在活跃的转录区域共转录招募的，这种联系部分依赖于Spt4和Spt6。对Elf1(Cid：1)突变体的分析表明，该因子在活跃转录区域维持适当的染色质结构方面发挥了作用。最后，ElF1的Puriﬁ阳离子提示与酪蛋白激酶II有关，以前曾涉及到转录中的作用。综上所述，这些结果表明Elf1在转录延伸的调节中发挥了重要作用。DST1(Cid：1)和URA3(Cid：1)基因。然后用mTn3转座子插入文库(59)中的NotI产生的线性DNA片段转化该菌株，并在缺乏亮氨酸的培养基上筛选转化子。将大约20,000个转化子复制到不含亮氨酸和含有5-FOA的培养液中。最初，由于在含有5-FOA的培养基上缺乏生长，28个合成致死候选被鉴定为ﬁ。对这些克隆进行纯化，并在5-ﬁ上重新测试其生长情况。由于转座子插入的不稳定性、复查时表型较弱或表型与转座子插入之间缺乏连锁，许多插入被排除为候选。在这些候选基因中，我们发现了一个强大的合成致死候选基因，其中表型与转座子插入有关。我们用载体ﬁ(34，56)鉴定了该插入片段的基因组位置。该插入片段位于YKL160W/ELF1基因中，预示着转座子插入的ﬁrst 62氨基酸与lacZ基因的融合。SGA分析。用少量Modiﬁ阳离子进行如上所述的SGA分析(74)。将酵母缺失集从96孔容器中的冻存转移到大的YPD平板上，在30℃下斑点生长2天，然后将菌株与查询菌株L1095的草坪在新鲜的YPD平板上交配，培养1天，然后复制模板到含有G418和clonNAT的YPD平板上进行二倍体筛选。将二倍体复制到产孢剂上，在22℃下培养5天。用复制法在含卡那瓦宁(50 mg/L)的SC-His-Arg平板上培养2天。最后，将菌株复制到含有G418的单倍体选择培养基和含有G418和clonNAT的单倍体选择培养基中。对生长进行3天的评分，并用与相同缓冲液平衡的钙调蛋白微珠(Pharmacia)识别合成致死候选ﬁ。在4℃下结合1~2 h后，用200(Cid：6)L钙调素结合缓冲液和200(Cid：6)L钙调素洗涤缓冲液(10 mM Tris-Cl，pH 7.9，10 mM(Cid：9)-巯基乙醇，0.1 mM CaCl2，0.1%Triton X-100,100 mMNaC)洗涤。用5(Cid：10)100(Cid：6)L钙调素洗脱缓冲液(10 mM Tris-Cl，pH 7.9，10 mM(Cid：9)-巯基乙醇，3 mM EGTA，pH 8.0，0.1%Triton X-100,100 mMﬁ)洗脱纯化的钙调蛋白复合体。纯化的ﬁ蛋白经直接还原、烷基化、胰酶消化后，在Finnigan LCQ Deca串联质谱仪上进行LC-MS/MS分析，或在含有10%聚丙烯酰胺的凝胶上用聚丙烯酰胺凝胶电泳法分离，然后用银染。蛋白条带还原、烷基化、胰酶消化后，将纯化的多肽以(ﬁ：5)-氰基-4-羟基肉桂酸(FLOKA)为基质进行斑点分析。基质辅助激光解吸电离-飞行时间(MALDITOF)在阳离子ReﬂEtron(39，63)模式下用Reﬂex IV(Bruker Daltonics，Bilerica，MA)仪器进行了分析。三氯乙酸沉淀的蛋白质再悬浮在100mMNH4HCO3-1mMCaCl2缓冲液中，pH 8.5，在37℃下与固定化Poros的2(CID：6)L消化过夜

In order to identify previously unknown transcription elongation factors, a genetic screen was carried out to identify mutations that cause lethality when combined with mutations in the genes encoding the elongation factors TFIIS and Spt6. This screen identiﬁed a mutation in YKL160W, hereafter named ELF1 ( el ongation f actor 1). Further analysis identiﬁed synthetic lethality between an elf1 (cid:1) mutation and mutations in genes encoding several known elongation factors, including Spt4, Spt5, Spt6, and members of the Paf1 complex. Genome-wide synthetic lethality studies conﬁrmed that elf1 (cid:1) speciﬁcally interacts with mutations in genes affecting transcription elongation. Chromatin immunoprecipitation experiments show that Elf1 is cotranscrip- tionally recruited over actively transcribed regions and that this association is partially dependent on Spt4 and Spt6. Analysis of elf1 (cid:1) mutants suggests a role for this factor in maintaining proper chromatin structure in regions of active transcription. Finally, puriﬁcation of Elf1 suggests an association with casein kinase II, previously implicated in roles in transcription. Together, these results suggest an important role for Elf1 in the regulation of transcription elongation. DST1 (cid:1) and the URA3 (cid:1) genes. This strain was then transformed with NotI-generated linear DNA fragments from an mTn3 transposon insertion library (59), and transformants were selected on medium lacking leucine. Ap- proximately 20,000 transformants were replica plated to medium lacking leucine and medium containing 5-FOA. Initially, 28 synthetic lethal candidates were identiﬁed due to the lack of growth on 5-FOA-containing medium. These colo-nies were puriﬁed and retested for growth on 5-FOA. Many of the insertions were eliminated as candidates due to the instability of the transposon insertion, weakness of phenotype upon rechecking, or lack of linkage between the pheno- type and the transposon insertion. Of these candidates, we found one strong synthetic lethal candidate where the phenotype was linked to the transposon insertion. We identiﬁed the genomic location of this insertion by vectorette PCR (34, 56). This insertion was located in the YKL160W/ ELF1 gene and predicts a fusion of the ﬁrst 62 amino acids with the lacZ gene from the transposon insertion. SGA analysis. SGA analysis was performed as previously described (74) with minor modiﬁcations. The S. cerevisiae deletion set was transferred from frozen stocks in 96-well containers to large YPD plates and grown as spots for 2 days at 30°C. The strains were then mated to lawns of the query strain L1095 on fresh YPD plates, incubated for 1 day, and then replica plated to YPD plates con- taining G418 and clonNAT to select for diploids. The diploids were replica plated to sporulation medium and incubated at 22°C for 5 days. MAT a spores were selected by replica plating to SC-his-arg plates containing canavanine (50 mg/liter) for 2 days. Finally, strains were replica plated to haploid selection medium containing G418 and haploid selection medium containing G418 and clonNAT. Growth was scored for 3 days, and synthetic lethal candidates were identiﬁed by calmodulin beads (Pharmacia) equilibrated with the same buffer. After binding for 1 to 2 h at4°C, the calmodulin beads were washed with 200 (cid:6) l calmodulin binding buffer and 200 (cid:6) l calmodulin wash buffer (10 mM Tris-Cl, pH 7.9, 10 mM (cid:9) -mercaptoethanol, 0.1 mM CaCl 2 , 0.1% Triton X-100, 100 mM NaCl). The puriﬁed protein complexes were eluted from the calmodulin beads with 5 (cid:10) 100 (cid:6) l calmodulin elution buffer (10 mM Tris-Cl, pH 7.9, 10 mM (cid:9) -mercaptoethanol, 3 mM EGTA, pH 8.0, 0.1% Triton X-100, 100 mM NaCl). The puriﬁed proteins were either directly reduced, alkylated, digested with trypsin, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on a Finnigan LCQ Deca tandem mass spectrometer or else separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on gels containing 10% polyacrylamide and stained with silver. After SDS-PAGE, protein bands were reduced, alkylated, and digested with trypsin, and puriﬁed peptide samples were spotted onto a target plate with a matrix of (cid:5) -cyano-4-hydroxycinnamic acid (Fluka). Matrix-assisted laser desorption ionization–time of ﬂight (MALDI-TOF) mass spec- trometry analysis was conducted utilizing a Reﬂex IV (Bruker Daltonics, Bil-lerica, MA) instrument in positive ion reﬂectron mode (39, 63). For tandem mass spectrometry, the trichloroacetic acid-precipitated protein was resuspended in 100 mM NH 4 HCO 3 –1 mM CaCl 2 buffer, pH 8.5, and digested overnight at 37°C with 2 (cid:6) l of immobilized Poros

Identiﬁcation and Characterization of Elf1, a Conserved Transcription Elongation Factor in Saccharomyces cerevisiae

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