Related Experiment Video
Updated: May 22, 2026
09:26
DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
Published on: December 29, 2021
A short adaptive path from DNA to RNA polymerases
Christopher Cozens1, Vitor B Pinheiro, Alexandra Vaisman
1Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
Summary
A novel DNA polymerase mutation (E664K) reveals a new specificity checkpoint, enabling RNA synthesis and modified nucleic acid production for biotechnology applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- DNA polymerase specificity is crucial for genome stability and biotechnological uses.
- Current models emphasize active site geometry for specificity control.
Purpose of the Study:
- To identify novel determinants of DNA polymerase substrate specificity.
- To explore the potential of engineered polymerases for novel nucleic acid synthesis.
Main Methods:
- Site-directed mutagenesis of Thermococcus gorgonarius (Tgo) DNA polymerase.
- Assessing translesion synthesis and RNA synthesis capabilities.
- Kinetic analysis of nucleotide incorporation and primer-template binding.
Main Results:
- A mutation (E664K) in the thumb subdomain, distant from the active site, acts as a specificity checkpoint.
- This mutation, combined with an active site mutation (Y409G), converts Tgo DNA polymerase into an RNA polymerase.
- The engineered polymerase synthesizes various modified RNAs from diverse primer types.
Conclusions:
- A previously unknown postsynthetic determinant of polymerase substrate specificity has been identified.
- This discovery provides insights into the evolutionary path from DNA to RNA polymerases.
- The engineered polymerase has significant implications for synthesizing noncognate nucleic acid polymers.
Related Concept Videos
03:01The Replisome
DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
03:01The Replisome
DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
01:59Lagging Strand Synthesis
During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
01:59Lagging Strand Synthesis
During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
01:53Bacterial Transcription
RNA polymerase (RNAP) carries out DNA-dependent RNA synthesis in both bacteria and eukaryotes. Bacteria do not have a membrane-bound nucleus. So, transcription and translation occur simultaneously, on the same DNA template.
Transcription can be divided into three main stages, each involving distinct DNA sequences to guide the polymerase. These are:
Transcription can be divided into three main stages, each involving distinct DNA sequences to guide the polymerase. These are:
00:43Bacterial RNA Polymerase
Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...