这项工作得到了国家卫生研究院的资助, AG040191 和 AG054566 向 VML 提供了支持。这项研究是进行的, 而 VML 是一个遥远的研究赠款接受来自美国老龄研究联合会。

1. 提取制剂
<ol>
	<li>YPD 板上的单一菌落的冷冻储存的条纹酵母菌株 (1% 酵母提取物、2% 蛋白胨、2% 葡萄糖和2% 琼脂)。在30° c 下孵育2天。</li>
	<li>接种酵母从 YPD 板 (使用一个单一的殖民地) 到15毫升的 YPD 培养基 (1% 酵母提取物, 2% 蛋白胨, 2% 葡萄糖) 在50毫升圆锥离心管和成长过夜与震动 (200-250 rpm) 在30° c。</li>
	<li>在2个无菌烧瓶中稀释培养成500毫升的 YPD 培养基, 使 OD600和 #60; 0.1。将酵母细胞在30° c 的震动下生长3-5 小时, 直至 OD600 = 0.5 (mid-log 阶段)。</li>
	<li>通过使用玻璃支架过滤器组件过滤0.45 μ m 膜过滤器来收集细胞。用刮刀刮出颗粒, 在液氮中闪冻, 在-80 ° c 处贮存。冷冻颗粒的预期尺寸是 ~ 0.2-0.5 克。 注意: 液氮温度极低;穿戴适当的保护。</li>
	<li>准备一个新的裂解缓冲液 (20 毫米的三 HCl pH 8.0, 140 毫米氯化钾, 5 毫米氯化镁2, 100 微克/毫升放线菌, 0.5 毫米的地面广播, 1% 海卫 X-100) 立即使用前, 保持在冰上。</li>
	<li>将3镀铬钢珠 (3.2 毫米) 放入1.8 毫升不锈钢管。预冷在液氮和添加冰冻颗粒, 盖上硅橡胶帽。质样品由 cryogrinding 为十年代在4200转每分钟;重复10次。冷却管在液氮至少十年代在每圆之间。 注意: 始终保持样品的冷冻是很重要的。</li>
	<li>加入1毫升的裂解缓冲液, 拌匀移。转移到一个新的1.5 毫升管。</li>
	<li>离心机在 2万 x g 为5分钟在4° c。在冰上工作时, 将上清液转化为新的1.5 毫升管。将100μ l 的裂解液转移到新的1.5 毫升试管中, 用于聚 (a) mRNA 的分离。立即进行 RNA 隔离或闪光冻结在液氮中的管。测量剩余的裂解物的外径260 , 这将用于足迹提取, 并在液氮中冻结管的闪光。在-80 ° c 下储存裂解。</li>
</ol>
2. 足迹提取
<ol>
	<li>蔗糖梯度的制备
	<ol>
		<li>准备50%、40%、30%、20% 和10% 蔗糖在梯度缓冲液中的溶液 (20 毫米三盐酸 pH 8.0, 140 毫米氯化钾, 5 mm 氯化镁2, 100 微克/毫升放线菌, 0.5 毫米的电喷)。</li>
		<li>吸管2.2 毫升的准备50% 蔗糖缓冲到底部的13.2 毫升薄壁 polyallomer 管和冻结解决方案为10分钟的-80 ° c (图 2)。然后将2.2 毫升的40% 蔗糖缓冲液放在冰冻的50% 蔗糖缓冲液的顶部, 并在-80 ° c 下冷冻10分钟。按照同样的指示, 30% 层, 然后 20%, 最后10% 蔗糖缓冲在彼此之上。避免在分层时制造气泡。将渐变保持在-80 ° c 直到使用。蔗糖梯度应至少在实验前一天准备, 并贮存在-80 ° c。</li>
	</ol>
	</li>
	<li>核酸消化
	<ol>
		<li>在4° c 实验前的晚上解冻蔗糖梯度。</li>
		<li>在冰上融化细胞裂解 (足迹样本)。将含有 50 OD260单元的细胞裂解物转移到新的1.5 毫升管中, 并将新鲜的裂解缓冲液分到1毫升。将剩余的裂解液储存在-80 ° c。添加10μ l 核糖核酸 I (100 U/μ l), 并在室温下孵育1小时, 在学杂费脚跟旋转上轻轻转动。</li>
		<li>选)澄清样品在 2万 x g 为5分钟在4° c 和恢复上清入一个新的1.5 毫升管子。</li>
	</ol>
	</li>
	<li>离心
	<ol>
		<li>在10-50% 蔗糖梯度的顶端轻轻地将裂解样品层化。</li>
		<li>Ultracentrifuge 的 polyallomer 管在 21万 x g (3.5万 rpm) 在4° c 为 3 h 使用 SW-41 钛转子。</li>
	</ol>
	</li>
	<li>梯度分馏系统设置
	<ol>
		<li>打开组件, 并允许紫外线显示器预热至少30分钟。</li>
		<li>如果使用数字记录软件, 请在计算机上启动软件, 将图形的缩放限制设置为-0.01 和1。</li>
		<li>用大通溶液填充注射器 (20 毫米的三盐酸 pH 8.0, 140 毫米氯化钾, 5 毫米氯化镁2, 60% 蔗糖), 去除注射器和套管内的气泡。</li>
		<li>安装一个 ultracentrifuge 管充满无核糖核酸水。用导管刺穿管子, 直到看到两个黑色的标记。启动注射器泵在1毫升/分钟。</li>
		<li>当水流经流动单元时, 在紫外线监测器上按 "自动零" 按钮, 将基线调整到0。将紫外线监测器的灵敏度设置为 "2.0 AU"。在建立了稳定的基线后, 停止注射器泵。恢复追逐解决方案, 并删除 ultracentrifuge 管。</li>
	</ol>
	</li>
	<li>Monosome 隔离
	<ol>
		<li>安装和刺穿含有蔗糖梯度的 ultracentrifuge 管。启动注射器泵在1毫升/分钟, 收集1毫升分数监测一个254值。池分数代表 80S monosome 峰值成一个管, 保持在冰 (图 3)。</li>
		<li>一旦追逐解决方案到达流动细胞, 停止注射器泵。恢复追逐解决方案, 并删除 ultracentrifuge 管。从步骤2.5.1 开始, 对其余样品重复分馏。 注: 完成后, 用至少30毫升的无核糖核酸水清洗分馏系统。用温水彻底冲洗油管、注射器和所有可拆卸部件。</li>
		<li>过滤分数通过0.5 毫升离心过滤器 (100 kDa 分子) 在 1.2万 x g 为10分钟在4° c 并且摒弃流动, 重复直到容量是和 #60; 100 μ l。添加400μ l 释放缓冲液 (20 毫米三盐酸 pH 7.0, 2 毫米 EDTA, 40 U/毫升核糖核酸抑制剂)。由移混合, 在冰上孵育10分钟, 并将该装置转换成新的收集管。离心机在 1.2万 x g 为10分钟在4° c 和收集流动通过。</li>
	</ol>
	</li>
	<li>脚印碎片净化
	<ol>
		<li>将含有足迹的 RNA 片段转移到新的1.5 毫升管中, 加入20μ l 20% SDS (最终浓度为 1%), 由移混合。</li>
		<li>添加1量的酸性苯酚: 氯仿 (pH 值 4.5), 涡旋为十年代。 注意: 酸性苯酚: 氯仿是有毒的, 避免接触皮肤和吸入。</li>
		<li>热在65° c 为5分钟, 在冰上投入1分钟离心样品在 1.2万 x g 为 5 min 在4° c, 转移水相 (上部) 到新的1.5 毫升管子。</li>
		<li>沉淀足迹 RNA 片段通过增加1/10 容量 NaOAc (pH 5.5), 1/100 容量糖原 (10 毫克/毫升) 和2.5 容量100% 乙醇。</li></ol></li></ol>孵育在-20 ° c 至少1小时。
	
	

3. 聚 (A) mRNA 提取
<ol>
	<li>总 RNA 提取
	<ol>
		<li>解冻100μ l 分的细胞裂解液 (总 RNA样本) 在冰上, 添加300μ l 20 毫米的三 HCl 的 pH 值7.0 和20μ l 20% SDS (到最后浓度 1%), 混合由移。</li>
		<li>添加1体积 (400 μ l) 的酸性苯酚: 氯仿 (pH 值 4.5) 和漩涡十年代。</li>
		<li>热在65° c 为5分钟, 在冰上投入1分钟离心样品在 1.2万 x g 为 5 min 在4° c, 转移水相 (上部) 到新的1.5 毫升管子。</li>
		<li>进行第二次苯酚提取。加入1的酸性苯酚: 氯仿 (pH 4.5), 涡旋为十年代. 热在65° c 为 5 min, 在冰上放1分钟. 离心样品在 1.2万 x g 为5分钟在4° c, 转移水相到新的1.5 毫升管。</li>
		<li>沉淀的 RNA。为此增加1/10 容量 3M NaOAc (pH 5.5), 1/100 容量糖原 (10 毫克/毫升) 和2.5 容量100% 乙醇。孵育在-20 ° c 至少1小时。</li>
	</ol>
	</li>
	<li>聚 (A) mRNA 分离
	<ol>
		<li>离心 RNA 样品在 2万 x g 为30分钟在4° c, 去除乙醇。三十年代在最大转速下旋转, 除去其余的乙醇与凝胶加载尖端, 空气干燥的颗粒5分钟。</li>
		<li>从聚 (A) mRNA 分离试剂盒中溶解300μ l 的裂解/结合缓冲液中的颗粒。根据聚 (a) mrna 隔离试剂盒制造商的协议进行聚 (a) mrna 提取。</li>
		<li>沉淀的 mRNA 样本增加1/10 卷的3米 NaOAc (pH 5.5), 1/100 体积的糖原 (10 毫克/毫升), 2.5 卷的100% 乙醇。孵育在-20 ° c 至少1小时。</li>
	</ol>
	</li>
	<li>mRNA 碎片
	<ol>
		<li>离心的 mRNA 样本在 2万 x g 为30分钟, 在4° c, 除去乙醇。向下旋转30秒, 将其余的乙醇与凝胶加载的尖端和空气干燥的颗粒5分钟。</li>
		<li>重 mRNA 在18μ l 核糖核酸的水中, 加入2μ l 的 10x RNA 碎片缓冲。在94孵育5分钟。把管子转到冰上</li>
		<li>沉淀通过增加1/10 容量 3 M NaOAc (pH 5.5), 1/100 容量糖原 (10 毫克/毫升) 和2.5 容量100% 乙醇。孵育在-20 ° c 至少1小时。</li>
	</ol>
	</li>
</ol>
4. 磷酸
<ol>
	<li>用 T4 核苷酸激酶治疗足迹和分裂的 mRNA 样本。为此, 离心样品在 2万 x g 为30分钟在4° c, 去除乙醇。三十年代在最大转速下降速, 用凝胶加载尖端除去乙醇的其余部分。空气干燥颗粒5分钟。</li>
	<li>重7.75 µL 水中的颗粒, 增加1µL 的 10x T4 核苷酸激酶缓冲, 1 µL 的 T4 核苷酸激酶 (1万 u/毫升), 和0.25 µL 的核糖核酸抑制剂 (20 u/µL)。在37° c 下孵育1小时。</li>
	<li>选)预运行15% 尿素凝胶在180伏, 15 分钟使用1x 缓冲。</li>
	<li>在每个脚印和 mRNA 样本中添加10µL 2X 尿素样品缓冲液至10µL。准备一个控制样本, 其中包含1µL 的10µM 上大小标记寡核苷酸 (32 nt), 1 µL 10 µM 低尺寸标记寡核苷酸 (28 nt), 8 µL 水, 和10µL 的2x 的尿素样本缓冲区的足迹样本。制备含有1µL 10 µM RNA 标记寡核苷酸 (63 nt), 9 µL 水, 10 µL 2x 的对照样品, 用于 mRNA 样品的尿素样品缓冲。</li>
	<li>孵育样品和控制3分钟在75° c, 旋转下来, 在冰上放1分钟. 将每个样品装入15% 个尿素凝胶的2井, 分离 RNA 片断由凝胶电泳在 180 V 为 1 h。</li>
	<li>用核酸凝胶染色 (在水中稀释 10,000x), 将凝胶染色5分钟, 免受光照。</li>
	<li>使用蓝色光源 transilluminator, 在28和 32 nt 标记之间切割合适的带区, 用于脚印样本 (图 4A) 和 50-70 nt, 用于 mRNA 样本 (图 4B), 使用干净的刀片。冷冻聚丙烯酰胺凝胶件在-80 ° c 至少10分钟。</li>
	<li>从聚丙烯酰胺凝胶中提取 RNA 如下。将凝胶件在70° c 处加热2分钟, 用一次性颗粒杵研磨凝胶。洗 RNA 与300µL 洗脱缓冲器 (20 毫米三 HCl pH 值 7.0, 2 毫米 EDTA), 增加1µL 核糖核酸抑制剂 (20 U 或µL) 和孵育在37° c 为 3 h。</li>
	<li>离心样品在 1.2万 x g 为5分钟在4° c, 收集上清, 并且沉淀通过增加1/10 容量 NaOAc (pH 5.5), 1/100 容量糖原 (10 毫克/毫升) 和2.5 容量100% 乙醇。孵育在-20 ° c 至少1小时。</li>
</ol>
5. 3 '-适配器结扎
<ol>
	<li>离心的足迹和 mRNA 样本在 2万 x g 为30分钟在4° c, 除去乙醇。三十年代在最大转速下降速, 用凝胶加载的尖端除去乙醇的其余部分。空气干燥颗粒10分钟。</li>
	<li>重4.75 µL 核酸水中的颗粒2µL 50% PEG8000, 1 µL 10X T4 rna 连接缓冲器, 1 µL 3 "-适配器 (100 ng/µL), 0.25 µL 核糖核酸抑制剂 (20 u/µL), 1 µL T4 rna 连接2截断 KQ (20万 u/毫升)。在16° c 的夜间孵育。</li>
	<li>第二天早上, 通过添加0.5 µL 5 "-deadenylase (10 u/µL) 和0.5 µL 的 Rec J 酶 (10 u/µL) 直接到结扎反应, 消除适配器的过剩。孵育30分钟, 在30° c。</li>
	<li>沉淀样品。为此, 添加30µL 核酸水, 1 µL 糖原 (10 毫克/毫升), 1/10 体积 NaOAc (pH 5.5), 2.5 卷100% 乙醇。孵育在-20 ° c 至少1小时。</li>
</ol>
6. 反向转录
<ol>
	<li>离心样品在 2万 x g 为30分钟在4° c, 去除乙醇。三十年代在最大转速下降速, 用凝胶加载的尖端除去乙醇的其余部分。空气干燥颗粒10分钟。</li>
	<li>重11.5 µL 核酸水中的颗粒, 加入0.5 µL 8 µM 反转转录底漆 (RT 底漆) 和1µL dNTP 混合 (10 毫米)。孵育5分钟在65° c, 在冰上的地方。</li>
	<li>添加4µL 的5X 第一股链缓冲, 2 µL 0.1 M 的µL, 0.5 核糖核酸抑制剂 (20 u/µL), 0.5 µL 反转录酶 (200 u/µL)。孵育30分钟, 在48° c, 1 分钟在65° c, 5 分钟在80° c。</li>
	<li>不沉淀和立即进行的水解 RNA, 增加0.8 µL 的2M 氢氧化钠, 孵化30分钟在98° c。添加0.8 µL 2M HCl, 以中和反应。</li>
	<li>沉淀样品。</li></ol>为此, 添加20µL 核酸水, 1 µL 糖原 (10 毫克/毫升), 1/10 体积 NaOAc (pH 5.5), 2.5 卷100% 乙醇。孵育在-20 ° c 至少1小时。
	<li>离心样品在 2万 x g 为30分钟在4° c, 去除乙醇。三十年代在最大转速下降速, 用凝胶加载的尖端除去乙醇的其余部分。空气干燥颗粒10分钟。</li>
	<li>重5µL 核酸水中的颗粒, 加入5µL 的2X 脲样缓冲液。孵育3分钟在75° c, 旋转下来, 在冰上放1分钟。</li>
	<li>选)预运行10% 尿素凝胶在180伏, 15 分钟使用1X 缓冲。</li>
	<li>在10% 尿素凝胶的1井上加载样品。用凝胶电泳将反转录反应产物分离为180伏50分钟。作为一个控制, 准备一个样品, 其中包含1µL 2.5 µM RT 底漆, 1 µL 2.5 µM 128 nt 标记寡核苷酸, 3 µL 水, 和5µL 2X 的尿素样本缓冲区, 并运行在一个单独的车道上的凝胶。</li>
	<li>用核酸凝胶染色 (在水中稀释 10,000x), 将凝胶染色5分钟, 免受光照。使用蓝色光 transilluminator, 用干净的刀片将 128 nt 和更高 (图 5) 的频带剪切。冷冻聚丙烯酰胺凝胶件在-80 ° c 至少10分钟。</li>
	<li>将凝胶件在70° c 处加热 2min, 用一次性颗粒杵研磨凝胶。洗 DNA 与300µL 的20毫米的三盐酸 pH 7.0, 并孵化在37° c 为 3 h。</li>
	<li>离心样品在 1.2万 x g, 为5分钟在4° c, 收集上清, 并且沉淀通过增加1/10 容量 NaOAc (pH 5.5), 1/100 容量糖原 (10 毫克/毫升) 和2.5 容量100% 乙醇。孵育在-20 ° c 至少1小时。</li>

7. Circularization
<ol>
	<li>离心样品在 2万 x g 为30分钟在4° c, 去除乙醇。三十年代在最大转速下旋转, 除去其余的乙醇与凝胶加载尖端, 空气干燥的颗粒10分钟。</li>
	<li>重16.75 µL 核酸水中的颗粒。添加2µL 的10x 单 DNA (ssDNA) 连接缓冲器, 1 µL 50 mM MnCl2, 0.25 µL ssDNA 连接 (100 U/µL)。孵育1小时, 在60° c, 10 分钟在80° c。不沉淀, 并贮存在-20 ° c 的 ssDNA 结扎反应产物。</li>
</ol>
8. PCR 文库扩增
<ol>
	<li>在冰上工作的时候在 PCR 管中混合如下: 146 µL 核酸水, 2 µL 20 µM 前引物, 2 µL 20 µM 索引反向底漆, 40 µL 5x 高保真 DNA plymerase 缓冲器, 4 µL dNTPs (10 mM), 4 µL ssDNA 结扎反应产物, 2 µL 高保真 DNA 聚合酶。选择一个不同的索引反向底漆为每一个样品, 将多路复用排序。将 pcr 混合物中的50µL 分转化为4新的 pcr 管。</li>
	<li>使用以下设置来执行 PCR 放大 (8、10、12、14) 不同的周期数: 1分钟 at 98 ° c 初始变性 8到14周期: 十五年代在94° c 5 s 在55° c 十年代在65° c 2分钟在65° c 最后的引伸</li>
	<li>通过添加1/10 体积的3米 NaOAc (pH 5.5), 1/100 的糖原 (10 毫克/毫升), 和2.5 卷的100% 乙醇沉淀 PCR 产品。孵育在-20 ° c 至少1小时。</li>
	<li>离心样品在 2万 x g 为30分钟在4° c, 去除乙醇。三十年代在最大转速下旋转, 除去其余的乙醇与凝胶加载尖端, 空气干燥的颗粒。</li>
	<li>重8µL 核酸水中的颗粒, 加入2µL 变性5x 核酸样品缓冲液。在1井变性8% 的凝胶中加载样品。用凝胶电泳分离 DNA 产物, 在150伏35分钟。作为一个控制, 准备一个样本, 其中包含0.5 µL 10 bp DNA 阶梯 (1 µg/µL), 7.5 µL 水, 和2µL 的变性5x 核酸样本缓冲区, 并运行在一个单独的通道的凝胶。</li>
	<li>用核酸凝胶染色 (在水中稀释 10,000x), 将凝胶染色5分钟, 免受光照。使用蓝光 transilluminator, 削减约 150 bp 为足迹样本和大约 180 bp 的 mRNA 样本 (图 6) 与清洁刀片的波段。储存凝胶件在-80 ° c 至少10分钟。</li>
	<li>将凝胶件在70° c 处加热2分钟, 用一次性颗粒杵研磨凝胶。洗 DNA 与300µL 的20毫米的三盐酸 pH 7.0, 并孵化在37° c 为 3 h。</li>
	<li>离心样品在 1.2万 x g, 为5分钟在4° c, 收集上清, 并且沉淀通过增加1/10 容量 NaOAc (pH 5.5), 1/100 容量糖原 (10 毫克/毫升) 和2.5 容量100% 乙醇。孵育在-20 ° c 至少1小时。</li>
	<li>离心样品在 2万 x g 为30分钟在4° c, 去除乙醇。三十年代在最大转速下旋转, 除去其余的乙醇与凝胶加载尖端, 空气干燥的颗粒。最后, 重了20µL 的核酸水库, 并进行了量化、质量控制和排序。</li>
</ol>
9. 图书馆量化和高通量测序
<ol>
	<li>在发送样本进行排序之前, 确定 PCR 扩增库的产量和质量。足迹和 mRNA 排序库的代表性配置文件如图 7所示。PCR 扩增文库的预期规模是 148-152 bp 用于足迹样本, 170-190 bp 用于 mRNA 样本。</li>
	<li>如果几个不同的条形码样本将被汇集在一起进行排序, 使用 qPCR-based 测序库定量化方法对库进行精确的量化。</li>
	<li>以摩尔比率汇集图书馆。计算池的总体积为3µL x 总数量的样品要汇集。确定需要添加的每个库的容量, 以便库在摩尔比率中混合, 以达到池的 10 nM 最终浓度。将每个库的计算量添加到一个新的1.5 毫升管, 由移混合。加水以达到计算总容积。在-20 ° c 处存储汇集库。</li>
	<li>在公司测序平台上运行 50 bp 单测序, 发送一个分的汇集库的排序。我们在一个单一的测序运行中例行地复用12样本, 每测序车道产生 ~ 2亿读数。 注意: 每个样本收集的足迹读数的最终数量取决于输入材料的数量和 rRNA 污染的程度。</li>
</ol>

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作者声明他们没有竞争的金融利益。

Ribo-Seq 方法已成为一个强大的技术, 分析 mRNA 翻译在体内在全基因组的水平3。使用这种方法进行的研究, 允许 single-codon 的分辨率进行监测, 有助于我们理解翻译规则。尽管有其优点, Ribo 还是有几个局限性。核糖体 RNA (rRNA) 片段总是 co-purified 在核糖体保护的足迹的分离, 减少了有用的测序读数, 可以获得在 Ribo-Seq 实验9,25的产量。...

mRNA 翻译是细胞中的基本过程之一, 在蛋白质表达调控中起着重要的作用。因此, mRNA 翻译受到严格控制, 以响应不同的内外生理刺激1,2。然而, 翻译规则的机制仍然理解。在这里, 我们描述了协议的基因组全定量翻译在萌芽酵母核糖体剖面。核糖体剖面技术的总体目标是研究和量化不同细胞条件下的特定基因的翻译。这项技术使用下一代测序来定量分析核糖体在整个基因组中的占有情况, 并允许监测蛋白质合成的速率在体内在单一密码子解析3,4。目前, 这种方法提供了最先进的测量蛋白质翻译水平的手段, 并已证明是一个有用的发现工具提供的信息, 不能透露其他现有的技术, 如微阵列或转换状态数组分析 (TSAA) 5。由于核糖体分析报告的转录水平和翻译输出的组合变化, 它也提供了比其他方法更大的灵敏度。
这种方法是基于核糖体保护的 mRNA 片段3的深度测序。在蛋白质翻译期间, 核糖体保护 ~ 28 nt 部分的 mRNA (称为脚印) 6。通过确定核糖体保护片段的序列, Ribo-Seq 可以映射核糖体在翻译的 mrna 上的位置, 并确定哪些区域的 mrna 可能被积极转化为蛋白质3,7。此外, 我们可以定量测量 mrna 的翻译, 通过计算与给定的 mrna 转录值对齐的脚印数。
为了分离核糖体保护的碎片, 细胞裂解最初处理的翻译抑制剂, 以拖住核糖体后, 核糖核酸消化。虽然游离的 mrna 和部分的翻译基因不受核糖体的保护, 退化的核糖核酸, 核糖体保护的 mrna 片段可以恢复纯化完整的核糖体足迹复合物。然后将这些 mRNA 的足迹转化为 cDNA 文库, 并通过深度测序 (图 1) 进行分析。在核糖体剖面的同时, 完整的 mRNA 从相同的样本中提取并测序。通过比较 Ribo-Seq 和 mrna 丰度测量的翻译水平, 我们可以确定在翻译水平上具体上调或下调的基因, 并在全基因组水平上计算 mrna 的翻译效率。虽然本文中描述的协议是针对酵母的, 但对于那些试图在其他系统中建立 Ribo 的研究人员来说, 它也应该是有用的。

<table>
	<tbody>
		<tr>
			<td>0.45 &#956;M membrane filters</td>
			<td>Millipore</td>
			<td>HVLP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9261</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL centrifugal filters (100 kDa MWCO)</td>
			<td>Millipore</td>
			<td>UFC510024</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.0</td>
			<td>Invitrogen</td>
			<td>AM9850G</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.5 (pH 8.0 at 4&#176;C)</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td></td>
		</tr>
		<tr>
			<td>10X TBE buffer</td>
			<td>Invitrogen</td>
			<td>AM9863</td>
			<td></td>
		</tr>
		<tr>
			<td>10% TBE-urea gel</td>
			<td>Invitrogen</td>
			<td>EC6875BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>15% TBE-urea gel</td>
			<td>Invitrogen</td>
			<td>EC6885BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2 M MgCl2</td>
			<td>RPI</td>
			<td>M24500-10.0</td>
			<td></td>
		</tr>
		<tr>
			<td>2X TBE-urea sample buffer</td>
			<td>Invitrogen</td>
			<td>LC6876</td>
			<td></td>
		</tr>
		<tr>
			<td>3M NaOAc, pH 5.5</td>
			<td>Invitrogen</td>
			<td>AM9740</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#39; Deadenylase (10 U/&#956;L)</td>
			<td>Epicentre</td>
			<td>DA11101K</td>
			<td></td>
		</tr>
		<tr>
			<td>5X Nucleic acid sample loading buffer</td>
			<td>Bio-Rad</td>
			<td>161-0767</td>
			<td></td>
		</tr>
		<tr>
			<td>8% TBE gel</td>
			<td>Invitrogen</td>
			<td>EC6215BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td>Invitrogen</td>
			<td>AM9722</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue light transilluminator</td>
			<td>Clare Chemical Research</td>
			<td>DR-46B</td>
			<td></td>
		</tr>
		<tr>
			<td>Chrome-steel beads, 3.2 mm</td>
			<td>BioSpec Products</td>
			<td>11079132c</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogrinder</td>
			<td>Biospec product</td>
			<td>3110BX</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>RPI</td>
			<td>C81040-5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition System</td>
			<td>DATAQ Instruments</td>
			<td>DI-245</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) solution mix (10 mM)</td>
			<td>NEB</td>
			<td>N0447L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Invitrogen</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient fractionation system</td>
			<td>Brandel</td>
			<td>BR-184X</td>
			<td></td>
		</tr>
		<tr>
			<td>High-fidelity DNA polymerase (2,000 U/mL)</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td>Supplied with 5X Phusion HF Buffer</td>
		</tr>
		<tr>
			<td>Next-generation sequencing library quantification kit</td>
			<td>Kapa Biosystems</td>
			<td>KK4824</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleic acid gel stain</td>
			<td>Invitrogen</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XE-90 ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) mRNA isolation kit</td>
			<td>Invitrogen</td>
			<td>61011</td>
			<td></td>
		</tr>
		<tr>
			<td>Rec J exonuclease (10 U/&#956;L)</td>
			<td>Epicentre</td>
			<td>RJ411250</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse transcriptase (200 U/&#956;L)</td>
			<td>Invitrogen</td>
			<td>18080093</td>
			<td>Supplied with 5X first-strand buffer and 0.1 M DTT</td>
		</tr>
		<tr>
			<td>RNA fragmentation buffer</td>
			<td>NEB</td>
			<td>E6186A</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase I (100 U/&#956;L)</td>
			<td>Invitrogen</td>
			<td>AM2295</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase inhibitor (20 U/&#956;L)</td>
			<td>Invitrogen</td>
			<td>AM2696</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone rubber caps</td>
			<td>BioSpec Products</td>
			<td>2008</td>
			<td></td>
		</tr>
		<tr>
			<td>ssDNA ligase (100 U/&#956;L)</td>
			<td>Epicentre</td>
			<td>CL9021K</td>
			<td>Supplied with 10X CircLigase II buffer and 50 mM MnCl2</td>
		</tr>
		<tr>
			<td>Stainless steel microvials, 1.8 mL</td>
			<td>BioSpec Products</td>
			<td>2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060-5000.0</td>
			<td></td>
		</tr>
		<tr>
			<td>SW-41 Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Pump Systems</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase (10,000 U/mL)</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td>Supplied with 10X T4 polynucleotide kinase buffer</td>
		</tr>
		<tr>
			<td>T4 RNA ligase 2 truncated KQ (200,000 U/mL)</td>
			<td>NEB</td>
			<td>M0373S</td>
			<td>Supplied with 10X T4 RNA ligase buffer and 50% PEG8000</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Bio-Rad</td>
			<td>1851148</td>
			<td></td>
		</tr>
		<tr>
			<td>Thinwall polyallomer tubes, 13.2 mL</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>UV monitor</td>
			<td>Bio-Rad</td>
			<td>7318160</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharomyces cerevisiae strain BY4741</td>
			<td>Open Biosystems</td>
			<td>YSC1048</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide quantification of translation in budding yeast by ribosome profiling

mRNA 转化为蛋白质是一个复杂的过程, 涉及多个层次的调节。通常认为, mRNA 转录的变化反映了蛋白质合成的变化, 但许多例外情况已经观察到。最近, 一种叫做核糖体剖面 (或 Ribo-Seq) 的技术已经成为一种强有力的方法, 它允许识别, 精确度高, 将 mRNA 的区域转化为蛋白质, 并在全基因组水平上量化翻译。在这里, 我们提出了一个广义的协议的基因组全定量翻译使用 Ribo-Seq 在萌芽酵母。此外, 结合 Ribo 序列数据与 mrna 丰度测量, 使我们能够同时量化的翻译效率数以千计的 mrna 转录在同一样本, 并比较这些参数的变化, 以响应实验操纵或在不同的生理状态。我们描述了一个详细的协议, 生成核糖体足迹使用核酸消化, 分离完整的核糖体足迹复合物通过蔗糖梯度分馏, 并准备 DNA 库的深度测序, 以及适当的确保准确分析体内翻译所需的质量控制。

详细的管道的生物分析核糖体的剖析数据已经描述了以前的8,9。此外, 几个研究小组已经开发了生物信息学工具的差异基因表达分析和处理的测序数据, 这是特定的核糖体分析方法10,11,12 ,13,14,<sup cla...

Watch this Scientific Journal Video about Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling at JoVE.com

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

翻译调控在蛋白质丰度控制中起着重要的作用。在这里, 我们描述一个高通量的方法, 定量分析翻译在萌芽酵母酿酒酵母。

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

genome-wide-quantification-translation-budding-yeast-ribosome

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

11.4K 次观看.  Boston University School of Medicine. 翻译调控在蛋白质丰度控制中起着重要的作用。在这里, 我们描述一个高通量的方法, 定量分析翻译在萌芽酵母酿酒酵母。

视频: Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

科研

生物化学

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA序列识别利用高通量原酶分析的DNAPrimase

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

双DNA标尺研究单核苷酸分辨率的核糖体易位机制

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

通过低温BN-MS分析进行高分辨率复杂分析

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

分析阿拉伯恐惧症中渗透应激信号的磷蛋白分析策略

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.