T7 RNA聚合酶转录与5位修饰的UTP衍生物
Jonathan D Vaught1, Torin Dewey, Bruce E Eaton
1College of Physical and Mathematical Sciences, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
Journal of the American Chemical Society
|September 10, 2004
概括
改性尿素三酸盐 (UTP) 衍生物有效地支持T7RNA聚合酶 (T7RNAP) 转录,产生全长的转录. 动力学研究表明,疏水性UTP修改不会显著改变转录动力学,而充电性修改会增加基质亲和度要求.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 合成化学 合成化学
背景情况:
- 未修改的尿素三酸盐 (UTP) 是RNA合成的基本基质.
- 对UTP的修改可以改变其属性,并将其纳入RNA.
- T7 RNA聚合酶 (T7 RNAP) 是一种用于体外转录的广泛使用的酶.
研究的目的:
- 评估7种UTP衍生物,通过胺结合在5位被修饰,作为T7RNAP转录的基质.
- 评估这些修改对转录产量和动力学的影响.
- 为了确定修改基是否阻碍RNA延长.
主要方法:
- 合成了7种具有多种5位胺基修饰的UTP衍生物.
- 使用T7 RNAP和使用各种DNA模板修改的UTP基质进行转录分析.
- 动力分析以确定T7RNAP的迈克利斯常数 (K(m)) 和最大速度 (V(max)) 以修改的UTPs.
主要成果:
- 所有七种UTP衍生品都产生了大量的全长转录,即使使用具有挑战性的DNA模板.
- 疏水性5位修饰 (,,,,,) 显示K (m) 和V (max) 值与未修饰的UTP相比.
- 在5位的伊米达和氨基基修饰导致K (m) 值显著更高.
- 所有测试的UTP衍生物都被纳入转录,而不会阻碍RNA延长 (类似V(max)).
结论:
- 五种具有疏水性5位修饰的UTP衍生物是T7RNAP转录的有效基质.
- 带正电荷的5位修改增加了酶的基质亲和度要求.
- 对UTP的5位修改本身并不妨碍由T7RNAP延长RNA转录.
相关概念视频
02:01pre-mRNA Processing
In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
00:58Eukaryotic RNA Polymerases
RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
All three eukaryotic RNAPs require specific transcription factors, of which the...
All three eukaryotic RNAPs require specific transcription factors, of which the...
02:36Transfer RNA Synthesis
One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
02:42Transcription Attenuation in Prokaryotes
Transcriptional attenuation occurs when RNA transcription is prematurely terminated due to the formation of a terminator mRNA hairpin structure. Bacteria use these hairpins to regulate the transcription process and control the synthesis of several amino acids including histidine, lysine, threonine, and phenylalanine. Transcription attenuation takes place in the non-coding regions of mRNA.
There are several different mechanisms used to attenuate transcription. In ribosome mediated...
There are several different mechanisms used to attenuate transcription. In ribosome mediated...
01:35Pre-mRNA Processing: Modification of pre-mRNA Ends
In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...
02:36Transfer RNA Synthesis
One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...