法改会- V的。希望本文献给博士Adriel D.约翰逊，一个美妙的教授，其的首要任务是对学生的教育内存。我们想念你，Adriel。我们感谢杰奎琳莫雷诺，她在协助执行在这项研究中中所描述的实验。支持这项研究是从美国国家科学基金会，MCB - 0615390至CY提供赠款，并到LRC - V提供的启动资金。在亨茨维尔的阿拉巴马大学。

1。无细胞提取物的制备。 <ol><li>两克菌体干大肠杆菌获得日志中期阶段文化洗净，两倍半公升的缓冲，一个含有10毫米的Tris -醋酸，pH值8.0，醋酸镁14毫米，60毫米醋酸钾，和50微克/毫升phenylmethanesulphonylfluoride（PMSF）5000 X克离心5分钟，在4 ° C。洗涤后，菌体悬浮于40 mL缓冲液A中</li><li>使用法国媒体细菌细胞被破坏，采用6000 psi的压力。这种压力相当于600个单位，使用一英寸活塞。中断悬挂收集在一个干净的玻璃管（50毫升）。 </li><li>被视为扰乱悬挂1毫米dithiotreitol（DTT），并在30,000 30分钟XG离心。离心过程是重复的，利用由此产生的上清液。最后上清分装成微管（1毫升）。最后，分装使用干冰乙醇的混合物或液氮冷冻和储存在-60 ° C或-80 ° C。 </li></ol> 2。 RF2贫的无细胞提取物的制备。 <ol><li> RF2在室温下使用一个旋转的轮子，一小时，反抗血清5毫升4.5毫升的蛋白质一个琼脂糖4B浆珠混合。离心混合物是在2500 XG从抗血清中分离出来的珠子。后丢弃上清液，这些珠子含有反RF2抗体（抗- RF2珠）后洗净由再悬浮在1毫升缓冲液B含有35毫米的Tris -醋酸pH值7.8，10毫米醋酸镁，30毫米醋酸铵，60 MM钾谷氨酸和5微克/毫升的亮肽素蛋白酶抑制剂。反RF2珠，然后使用如上所述的相同条件下离心分离缓冲液B。洗涤过程重复两次。 </li><li>一毫升的无细胞提取物混合150μL反RF2珠使用一个旋转的轮子，在4 ° C，为两个小时。万XG混合物离心分离从珠无细胞提取物。上清拆除和处理又与另一反RF2珠150μL。由此造成的无细胞提取液，此过程重复一次。最后的无细胞提取液配发到微管（100μL）。分装是使用干冰乙醇的混合物或液氮冻结，并储存在-60 ° C或-80 ° C。 </li></ol> 3。制备的DNA模板。 <ol><li>准备1毫升混合包含要翻译的序列的质粒模板（图1）600 femtomoles PCR反应，每个图1中，每一个dNTP的0.2微摩尔和50的U表示的寡核苷酸与0.4纳摩尔的Taq Stratagene公司有限公司提供的缓冲区中的DNA聚合酶执行后续条件下的扩增反应：94℃2分钟，（94 ° 55 ° C 30秒，30秒彗星，72 ° C为1分; 30周期），72 ° C下12分钟。 </li><li>加入1 / 10体积的3 M醋酸钠，pH值5.2，和2卷冰冷的乙醇沉淀的DNA纯化的PCR产品。重复一次降水过程。重悬于100μL二乙基焦碳（DEPC）处理水的DNA。 通常情况下，此过程会产生一个600 bp的DNA产品100微克。 </li><li>验证琼脂糖凝胶电泳PCR产物的完整性。 </li></ol> 4。制备生物素标记的mRNA。 <ol><li>准备在体外转录反应DEPC处理水，搅拌5微克的PCR产生的DNA片段与0.5微摩尔的ATP，CTP，GTP，0.3微摩尔的生物素- 16 - UTP双绞线，50纳摩尔，和10μL，100 μL， Promega公司提供的缓冲区T7酶混合反应混合物在37 ° C为3小时。 为了量化获得表达量，应该被淘汰的DNA为模板，加入无RNase的DNA酶。孵育反应在37℃10分钟。 </li><li>净化沉淀的RNA，加入1 / 10体积3 M醋酸钠，pH值5.2，冷乙醇和2卷的mRNA的产品。重复一次降水过程。悬浮在100μLDEPC处理水的mRNA。 通常，此过程产生的600 NT素1-2毫克之间标记基因的产品。 </li><li>琼脂糖凝胶电泳验证了生物素标记的mRNA的产品的完整性。 </li></ol> 5。含肽酰- tRNA的翻译核糖体的分离。 <ol><li>准备500μL的DEPC处理水体外翻译反应混合物搅拌10-15微克生物素标记的基因与75纳摩尔每十九氨基酸以外的所有氨基酸，将改为由放射性氨基酸，50μCi的放射性氨基酸（37MBq），和20-5<html> 0 RF2 -耗尽无细胞提取物在缓冲反应混合物含有pH值8.0的Tris -醋酸，40毫米，10毫米醋酸镁，醋酸钾175毫米，10毫米醋酸铵，数码地面电视的2毫米，2毫米的ATP，0.5μL的，毫米GTP，30毫米磷酸（PEP），0.3 U / mL的丙酮酸激酶（PK），3.5％聚乙二醇8000，1毫米的精胺，20μg/ mL的叶酸和250微克/毫升的 tRNA从E 大肠杆菌 。反应混合物在37℃，10分钟。 除了反应元件的顺序是非常重要的。混合无细胞提取物，必须先用缓冲液中的氨基酸和最终的混合物在室温下5分钟孵育允许核糖体的激活解决方案。后来，加入生物素标记的mRNA。对于使用化学改性过程的结构分析，增加了放射性氨基酸并不是必需的。 </li><li>添加到翻译反应混合物 - 3毫升顺磁性悬浮在缓冲液C含有pH值8.0的Tris -醋酸，35毫米，10毫米醋酸镁，醋酸钾175毫米，10毫米醋酸铵和1毫米的DTT和素珠（SMB）。新的悬挂在室温下孵育10分钟。 </li><li>应用磁场（MF），利用磁选站，从混合物分离的中小企业。 </li><li>悬浮在缓冲液C的SMB和单独的珠子，再次使用的MF。重复此洗涤过程两次。在500μL缓冲液C中重悬珠，储存在冰暂停，并立即执行下一个步骤。 </li></ol> 6。隔离含有肽酰- tRNA的核糖体的分析。 对于肽酰- tRNA <ol><li>混合使用10μL含50毫米的Tris - HCl，pH为6.8，2.5％（V / V）甘油，4％的十二烷基硫酸钠，2毫米数码地面电视和0.2毫克的缓冲区装载10μL的SMB（悬浮在缓冲液C） / mL的溴酚蓝。解决运行10％的Tris -甘氨酸聚丙烯酰胺凝胶样本珠的组件。 </li><li>使用真空凝胶干燥机，干凝胶。 </li><li>验证由一个X光片露出了干凝胶的肽酰- tRNA的完整性和净化。 </li></ol>核糖体RNA <ol start="4"><li> 2毫米乙二胺四乙酸（EDTA）解决方案，准备用DEPC处理过的的水，加入190μL和200μL苯酚与水平衡，以10μL珠悬挂。严厉震荡混合悬挂和无机相通过离心分离有机相在室温下3分钟在万XG。收集在一个新的微管上的水层。 </li><li>水层的RNA沉淀，加入1 / 10体积的3 M醋酸钠，pH值5.2，1μL20毫克/毫升的肝糖原的解决方案，冰冷的乙醇和2卷。在10μLDEPC处理过的水重悬的RNA。 </li><li>验证琼脂糖凝胶电泳的核糖体RNA的完整性。 通常情况下，50珠悬浮液产生的核糖体RNA1μg。 </li></ol> 7。代表性的成果： 图2给出了一系列的评估的质量和使用的程序隔离的翻译核糖体的功能概述分析结果。聚丙烯酰胺凝胶中解决了一个独特的乐队的观察结果显示，势必生物素标记的SMB（图2A）mRNA的的多肽存在。核糖体RNA的纯化，使用此程序建立，以及绑定到这些生物素标记的mRNA（图2B）的核糖体的存在。除了 ​​对嘌呤霉素，抗生素，诱导核糖体肽基转移酶活性，在新生肽- tRNA的1乳沟的结果。这是观察在聚丙烯酰胺凝胶中分离出的多肽（图2C）的迁移模式的转变。总之，这些数据表明，生物素标记的连接到SMB的mRNA含有肽酰- tRNA的功能核糖体。 含有特定的肽- tRNA的翻译核糖体的隔离，允许肽基tRNA基因，抗生素，和其他分子的影响的研究，对核糖体的功能（图3A），核糖体的结构（图3B）。在这里介绍的例子抗生素sparsomycin和氨基酸色氨酸（Trp）抑制在孤立的核糖体（图3A）RF2新生tnaC - tRNA的肽酰- tRNA诱导水解断裂。 sparsomycin或色氨酸与核糖体的相互作用也导致一些核苷酸构成的翻译核糖体（图3B）的23S rRNA的结构变化。总之，这里描述的生物材料，获得使用过程中获得额外的参与抑制核糖体功能后，其与各种分子因素之间的相互作用的结构接触了解的是有用的。 ove_content“&gt; <img src="/files/ftp_upload/2498/2498fig1.jpg" alt="图1" /> 图1，用于生产和净化翻译核糖体含有肽酰- tRNA的的过程。 DNA片段CA。 650 bp的长度，包含tnaC基因及其邻近地区，如图所示，在准备的mRNA含有生物素，（生物）人民运动联盟]的mRNA作为模板。这些DNA片段是由使用在图上方指出的寡核苷酸的PCR扩增程序。下划线的字母标记在核苷酸引物编码的T7启动子的位置。粗体字母表示的服务Dalgarno序列tnaC mRNA序列的翻译。 T7启动子的DNA片段是公认的T7 RNA聚合酶是用来抄写（箭头）tnaC序列和非编码序列下游。终止时，RNA聚合酶到达后的rpoBC终止序列（rpoBC“T”）的3&#39; -末端的DNA片段，的转录。茎环结构的rpoBC终止保护的mRNA的3&#39; - 5&#39;的exoribonuclease活动目前使用的无细胞提取物在体外翻译反应。隔离（生物）人民运动联盟]的mRNA被用来产生停滞，翻译的核糖体， 在体外翻译实验中使用。 tnaC序列[（生物）人民运动联盟] mRNA的摊位被纳入最后的氨基酸时，由于没有释放因子（RF2）从反应混合物中核糖体翻译。核糖体（生物）人民运动联盟] mRNA的复合物从总的反应链霉亲和磁性珠（SMB）将生物素标记的mRNA中的核苷酸的混合物中分离。中小企业势必给核糖体[（生物）人民运动联盟]通过使用一个磁场（MF），从总的反应混合物中提取mRNA的复合物。 <img src="/files/ftp_upload/2498/2498fig2.jpg" alt="图2" /> 图2。孤立的复合物的结构和功能分析。 A） 在体外翻译反应与执行[（生物）人民运动联盟] tnaC基因的起始密码子是由一个终止密码子的mRNA 。最终产品的使用链霉亲和素磁珠分离，如在图1所示。反应产物和孤立分子，然后由聚丙烯酰胺凝胶电泳分析。 tnaC - tRNA和tnaC肽带标记。 b）总RNA的提取用酚的提取程序从停滞不前的复合物。每个分离出的RNA琼脂糖凝胶电泳解决。核糖体RNA（rRNAs）表示。三）隔离的复合物含有tnaC - tRNA的培养（+）或无（ - ）0.02毫米嘌呤（普罗）在室温为10分钟。 tnaC肽被打成期间使用[35S] -蛋氨酸（A）或[3H] -脯氨酸（二）翻译。 <img src="/files/ftp_upload/2498/2498fig3.jpg" alt="图3" /> 图3含有tnaC氨酰- tRNA绑定到的抗生素sparsomycin，或色氨酸的核糖体的结构和功能分析。一）含有tnaC - tRNA的隔离复合物孵育（+）或无（ - ）sparsomycin（SPAR）或色氨酸（TRP）在室温5分钟。后来，混合物的混合与RF2和孵育20分钟在37 ° C。二）在23S rRNA基因甲基化的改变分析诱导分子内的核糖体具有约束力。隔离的复合物预培养（+）或无（ - ）2毫米色氨酸或思达为5分钟，在37 ° C。然后，烷基化剂，硫酸二甲酯，是补充和混合物在室温下放置一个额外的20分钟。提取总RNA和引物延伸分析[32P标记的寡核苷酸互补的间距下游100核苷酸的修饰核苷酸的23S rRNA基因的核苷酸。扩展实验的最终产品聚丙烯酰胺凝胶电泳解决。色氨酸或思达存在影响的23S rRNA基因核苷酸表示。

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+translation+termination+at+the+upstream+open+reading+frame+in+s-adenosylmethionine+decarboxylase+mRNA.">Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.</a> J. Biol. Chem. 277, 5988-5994 (2002).</li><li>Cruz-Vera, L. R., Rajagopal, S., Squires, C., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+ribosome-peptidyl-tRNA+interactions+essential+for+tryptophan+induction+of+tna+operon+expression.">Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression.</a> Mol. Cell. 19, 333-343 (2005).</li><li>Cruz-Vera, L. R., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+Residues+Asp16+and+Pro24+of+tnaC-tRNAPro+Participate+in+Tryptophan+Induction+of+tna+Operon+Expression.">Conserved Residues Asp16 and Pro24 of tnaC-tRNAPro Participate in Tryptophan Induction of tna Operon Expression.</a> J. Bacteriol. 190, 4791-4797 (2008).</li><li>Cruz-Vera, L. R., Yang, R., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tryptophan+inhibits+Proteus+vulgaris+tnaC+leader+peptide+elongation,+activating+tna+operon+expression.">Tryptophan inhibits Proteus vulgaris tnaC leader peptide elongation, activating tna operon expression.</a> J Bacteriol. 191, 7001-7006 (2009).</li><li>Cruz-Vera, L. R., New, A., Squires, C., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+features+essential+for+tna+operon+induction:+tryptophan+binding+at+the+peptidyl+transferase+center.">Ribosomal features essential for tna operon induction: tryptophan binding at the peptidyl transferase center.</a> J. Bacteriol. 189, 3140-3146 (2007).</li><li>Shimizu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+translation+reconstituted+with+purified+components.">Cell-free translation reconstituted with purified components.</a> Nat. Biotechnol. 19, 751-755 (2001).</li><li>Kinoshita, Y., Nishigaki, K., Husimi, Y. F. l. u. o. r. e. s. c. e. n. c. e. -. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=isotope-+or+biotin-labeling+of+the+5+'-end+of+single-stranded+DNA/RNA+using+T4+RNA+ligase.">isotope- or biotin-labeling of the 5 '-end of single-stranded DNA/RNA using T4 RNA ligase.</a> Nucleic Acids Res. 25, 3747-3748 (1997).</li><li>Nishigaki, K., Taguchi, K., Kinoshita, Y., Aita, T., Husimi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-ligation:+an+efficient+method+for+ligating+single-stranded+DNAs+and+RNAs+with+T4+RNA+ligase.">Y-ligation: an efficient method for ligating single-stranded DNAs and RNAs with T4 RNA ligase.</a> Mol Divers. 4, 187-190 (1998).</li></ol>

这本报告中所述的分离过程是高度重现。不幸的是，从相同的翻译序列产生的短肽- tRNA的存在，不能方便地避免这样的肽- tRNA的可能对应到15-20％的总绝缘材料（图2C）。这些污染物的浓度可能会增加，较高浓度的生物素标记基因已被用于或时无细胞提取物的使用旧的或已重复使用多次。因此，它是非常重要的控制组件的质量和浓度在体外翻译反应。另外，高效率的商业重组的无细胞提取物可以用于同样的目的（纯系统）18 。 使用这种方法，生物素- 16 - UMP是随机纳入沿靶mRNA的长度。有一个可能性，可能有一些的ORF含有尿苷大片纳入这个修改过的核苷酸序列，影响他们的翻译的。 biotin-16-UTP/UTP变量的相对浓度测试，以获取最高效的翻译过程中的mRNA的合成。可以用生物素标记的替代方法，针对5&#39; -端，最稳定的mRNA结束。 （一）生物素可以连接到使用3&#39; - NH2ATP（3&#39; -氨基，3&#39; -脱氧腺苷三磷酸腺苷）和T4 RNA连接酶 19的mRNA的5 &#39;端。 （二）5&#39;端生物素标记的寡核苷酸，可连接使用T4 RNA连接酶的基因， 以及 20的5&#39;端。 （三）3&#39; -末端生物素标记的寡核苷酸对应的mRNA的5&#39;端的互补序列，也可以用来隔离翻译复合物。这些生物素标记的寡核苷酸，可以连接到SMB前隔离。后来的SMB连接生物素标记的寡核苷酸，可以允许与他们互补的mRNA序列互动的翻译反应混合物混合。隔离后，混合RNA - DNA区 - 和地区之间的生物素标记的寡核苷酸与mRNA序列杂交 - 核糖核酸酶H可降解，分离从链霉珠陷入僵局的复合物​​。这种替代方法可能会允许复合物，在未来的结构分析，使用更高分辨率的方法聘用。

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Tris base</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>252859</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium acetate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63049</td>
<td>&nbsp;</td>
<tr>
<td>Potassium glutamate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium acetate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>09688</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protein A sepharose 4B slurry beads</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9424</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptin inhibitor</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L9783</td>
<td>&nbsp;</td>
<tr>
<td>Taq-DNA polymerase</td>
<td>&nbsp;</td>
<td>Stratagene</td>
<td>201223</td>
<td>&nbsp;</td>
<tr>
<td>T7 Maxiprep kit</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>P1300</td>
<td>&nbsp;</td>
<tr>
<td>SMB</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>Z5482</td>
<td>&nbsp;</td>
<tr>
<td>Biotin-16-UTP</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1388908</td>
<td>&nbsp;</td>
<tr>
<td>ATP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>GTP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8877</td>
<td>&nbsp;</td>
<tr>
<td>PEP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7002</td>
<td>&nbsp;</td>
<tr>
<td>PK</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7768</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylenglycol 8000</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>81268</td>
<td>&nbsp;</td>
<tr>
<td>Folinic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>47612</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>85558</td>
<td>&nbsp;</td>
<tr>
<td>tRNA from E. coli</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R1753</td>
<td>&nbsp;</td>
<tr>
<td>DEPC</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5758</td>
<td>&nbsp;</td>
<tr>
<td>Tricine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5816</td>
<td>&nbsp;</td>
<tr>
<td>L-amino acids</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>LAA21</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>43815</td>
<td>&nbsp;</td>
<tr>
<td>magnetic separation stands</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>Z5342</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of translating ribosomes containing peptidyl-trnas for functional and structural analyses

近日，有详细的结构和生化研究许多在新生肽合成抗生素抑制蛋白质的合成过程中，发生在核糖体的分子事件。这些抗生素，大多在形成肽- tRNA的监管新生多肽，抑制要么肽键形成或翻译终止1-7。抑制这些事件可以阻止核糖体的运动，这种现象被称为“翻译逮捕”。抗生素或新生肽诱导的翻译逮捕已被证明，以规管在不同的细胞，如细 ​​胞生长，抗生素耐药性，蛋白质易位和细胞的新陈代谢8-13功能有关的基因的表达。如果我们要了解完整的核糖体在翻译的作用，在每一个有机体，抗生素和监管新生多肽如何改变核糖体的功能的知识是必不可少的。 在这里，我们描述了一个简单的方法，可用于净化，专门分析，这些核糖体的翻译一个特定的mRNA，并包含一个特定的肽酰- tRNA 14。这个程序是基于绑定到一个生物素标记的mRNA翻译的核糖体的选择性分离。这些翻译的复合物是分开相同的混合物中的其他核糖体，采用链霉亲顺磁性珠（SMB）和磁场（MF）。合成生物素标记的mRNA为模板，PCR产生的DNA片段，含有T7转录促销员决胜转录使用检测。 T7 RNA聚合酶结合生物素UTP生物素- 16 - UMP;我们的条件下，约10个生物素- 16 - UMP分子纳入600 NT 25％的人民运动联盟的内容表达。这些生物素标记的mRNA分离，并在体外翻译释放因子2（RF2）进行检测耗尽的大肠杆菌菌株，含有野生型或突变核糖体获得的无细胞提取物。核糖体翻译的生物素标记的mRNA序列停滞在终止密码子区域，由于RF2蛋白质，通常完成翻译终止的情况下。孤立和停滞不前的核糖体含有新合成的肽酰- tRNA从翻译使用SMB的中频反应中删除。这些珠子只能绑定含生物素的消息。 隔离，平移的复合物，可用于分析野生型或突变核糖体元件，或肽- tRNA的序列，以及确定用抗生素或其他分子因素1,14-16核糖体相互作用的结构和功能特性。为了研究这些孤立的核糖体复合体的功能，可以进行肽基转移酶检测在嘌呤霉素的抗生素1存在。要研究翻译复合物的结构变化，以及既定的程序可以使用，如i）交联特定的氨基酸 14和/或ii）烷基化保护分析 1,14,17 。

Watch this Scientific Journal Video about Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses at JoVE.com

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

含新生肽- tRNA的核糖体的生化分析的一个主要障碍已被其他核糖体的存在，在同一样品的核糖体不参与翻译的特定基因序列分析。我们制定了一个简单的方法，净化，完全包含利益的新生肽酰- tRNA，核糖​​体。

分离翻译含肽- tRNA的核糖体的功能和结构分析

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

isolation-translating-ribosomes-containing-peptidyl-trnas-for

Research

JoVE Journal

Biology

19.7K 次观看.  University of Alabama Huntsville. 含新生肽- tRNA的核糖体的生化分析的一个主要障碍已被其他核糖体的存在，在同一样品的核糖体不参与翻译的特定基因序列分析。我们制定了一个简单的方法，净化，完全包含利益的新生肽酰- tRNA，核糖​​体。

视频: Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

科研

生物学

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.