基础知识
转录起始点(TSS):转录时,mRNA链第一个核苷酸相对应DNA链上的碱基,通常为一个嘌呤。UTR(Untranslated Regions):即非翻译区,是信使RNA(mRNA)分子两端的非编码片段。
5′-UTR从mRNA起点的甲基化鸟嘌呤核苷酸帽延伸至AUG起始密码子,3′-UTR从编码区末端的终止密码子延伸至多聚A尾巴(Poly-A)的末端。
![](http://bioengx.hk1.91site.net/wp-content/uploads/2017/03/Picture1-1.png)
查找基因的启动子区域-NCBI
1. 打开PubMed:https://www.ncbi.nlm.nih.gov/pubmed
2. 选择Gene,输入IL17A,点击search,结果如下图,点击第一个:
3. 下拉到下图位置,可以看到该基因的以下信息:
![](http://bioengx.hk1.91site.net/wp-content/uploads/2017/03/Picture5.png)
还可以看到如下序列信息:
4. 以上只是该基因的一些信息,可以用于查找相应的UTR等区域,下面进入正题,寻找promoter区域。还是拉到如下图位置,点击FASTA:
5. 基因位置信息如下图:
6. 一般认为基因上游2 kb区域为该基因的promoter区域,所以将基因上游2 kb序列调出来:
7. 复制上述序列就是基因的启动子序列了。
查找基因的启动子区域-UCSC
![](http://bioengx.hk1.91site.net/wp-content/uploads/2017/03/Picture10.png)
![](http://bioengx.hk1.91site.net/wp-content/uploads/2017/03/Picture11.png)
![](http://bioengx.hk1.91site.net/wp-content/uploads/2017/03/Picture12.png)
![](http://bioengx.hk1.91site.net/wp-content/uploads/2017/03/Picture13.png)
5. 得到下面的序列信息,开头直到第一个大写字母前面的所有小写字母序列即为该基因的promoter序列,你可以跟NCBI上得到的序列比对一下,看看是不是一样的呢?
6. 当然查找promoter的网站有很多,比如UCSC,在这里就不介绍了,大家可以自行探索,或者加小编微信amateur_1988交流。
转录因子结合位点的预测![](data:image/png;base64,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)
1. 打开http://jaspar.genereg.net/(我这边这个网址暂时打不开了,所以我登录了这个网址:http://jaspardev.genereg.net/),输入转录因子NFAT,点击Quick Search:
2. 将promoter序列粘贴进入右下角的框中,选中左侧转录因子,点击SCAN:
3. 得到28条转录因子NFAT与IL17A的结合位点,其中Strand -1没有特殊意义,只需选择Strand 1即可。
4. 好了,转录因子与promoter结合位点已经有了,接下来就是愉快的通过实验验证了!Luciferase、点突变、截短、ChIP等统统拉上来就可以了!
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