Question 1 - RNA polymerase II transcribes messenger RNA (mRNA). However, cells also need to
make other types of RNA that are essential for splicing the pre-mRNA (small nuclear; snRNA),
generating ribosome proteins for translation (ribosomal; rRNA) and translating the mRNA into
protein (transfer; tRNA). What RNA polymerases are responsible for the transcription of these other
types of RNA?
Sm-class snRNA—Pol Ⅱ（大多数）
；Lsm-class snRNA—Pol Ⅲ（少部分）
；
rRNA—Pol Ⅰ，转录起始还需要 SL1 和 UBF；
tRNA—Pol Ⅲ，启动子包含两个区域：box A&B；
Question 2 - Promoter regions in single-celled eukaryotes (i.e. yeast) contain a TATA-box for TATA
binding protein (TBP) to bind and start assembly of the transcription initiation complex. However,
in the multi-cellular eukaryote Drosophila only 43% of promoters contain a TATA-box. How do the
general transcription factors (GTFs) bind at genes that lack a TATA-box?
没有 TATA box，但是有 Sp1 结合位点。Sp1 属于 Sp/KLF 转录因子家族，含有锌指蛋白结构
域，能与 DNA 直接结合。
转录因子识别其他启动子序列，RNA 聚合酶与这些启动子序列结合。
Question 3 - In addition to the four main histones (H2A, H2B, H3 and H4) eukaryotic cells also
contain histone variants that have specific functional roles. What is the main functional role of the
H2A histone variant H2AX?
H2AX ADP 核糖基化在碱基切除修复（BER）中起着关键作用。
从机理上讲，E141 上的 ADP-核糖基化介导 Neil3 糖基化酶募集到 BER 的 DNA 损伤位点。
此外，这种 ADP 核糖基化的缺失会增强 DNA 氧化损伤时 H2AX（γH2AX）的丝氨酸 139
磷酸化，并错误地导致 DNA 双链断裂（DSB）反应因子的积累。
综上所述，这些结果表明，H2AX ADP 核糖基化不仅促进了 BER 修复，而且抑制了γH2AX
介导的 DSB 反应。
Question 4 - For transcription and duplication proteins need to bind to DNA at certain locations in
the genome. How do we know this? What techniques have been used to show protein-DNA
interactions and how do you determine the consensus sequence of DNA that a particular protein
binds to?
http://rs.yiigle.com/CN121382201902/1150280.htm
染色质免疫沉淀技术、甲基化干扰法、荧光技术、表面等离子共振技术、噬菌体展示技术；
电泳迁移率变动分析（electrophoretic mobility shift assay，EMSA）是用核酸探针和样本蛋白
混合孵育，样本中的核酸-蛋白质复合物在聚丙烯酰胺凝胶的迁移速度会慢于游离的探针并
形成一条滞后带，根据凝胶上滞后带的有无和多少来定量 DNA 结合蛋白的有无和结合活性
[3]。EMSA 是一种简单、快速、可定性与定量的体外蛋白质与 DNA 相互作用检测方法，且
适用于未经纯化的蛋白与纯化的重组蛋白[4]；其缺点是对蛋白复合体与 DNA 的结合无法鉴
定，难以比较不同片段间亲和力的差异，且不能真实的反映体内蛋白质与 DNA 相互作用的
生物过程。传统 EMSA 采用放射性核素标记寡核苷酸探针，会对环境和人体造成伤害故未
被广泛应用。Shi 等[5]使用改进的 EMSA 揭示与β-连环蛋白相互作用的血管内皮生长因子
（VEGF）的转录调节过程，其采用的化学发光法方便、快捷且非常敏感，克服了传统方法
的安全问题，使得该方法的应用更加普遍。
Question 5 - What sequence in the DNA signals for RNA polymerase II to stop transcribing a gene
and start adding a polyadenylation (poly-A) tail to the mRNA transcript?
Poly-A Signal，募集 CPSF 和 CSTF；
The principle of Phage display techniques (PDT) is to insert the gene to be selected into the gene of
the phage coat protein without affecting the function of the phage, so that the gene expression
products are displayed on the surface of the phage, and then react with the specific DNA fragment.
Phages with specific peptides or proteins can be screened by affinity enrichment to obtain binding
domain peptide or protein sequences that can bind to specific DNA.
PDT can effectively transfer genotypes and phenotypes in vitro, and its expressed products are
natural conformational proteins or peptides. It has the characteristics of fast, efficient, economical
and good applicability [28]. However, some sequences cannot be expressed in phage, and the
transformation packaging during phage display limits the capacity and molecular diversity of the
library.