A reverse transcription reaction allows the production of complementary DNA
(cDNA) using an RNA template and relies on polymerases displaying reverse
transcriptase (RT) activity. This process, with major applications in both
research and in medical diagnostics, is often limited by the nature of the RTs
available. RNA secondary structure can prove problematic where mesophilic
retroviral RTs are used while the alternative approach, using thermophilic DNA
polymerases with RT activity, often results in error-prone cDNA production.
This project recognised the need to study other possible sources of thermophilic
RTs and outlines the study of four previously uncharacterised Group II Intronencoded
proteins (IEP), with RT domains, from thermophilic bacteria. While
cloning of the IEP genes and their expression on a small scale proved
successful, difficulties were encountered when attempting purification. Despite
a lack of overall purity, samples containing IEPs from Thermosinus
carboxydivorans and Petrotoga mobilis were shown to have RT activity but
characterisation of these IEPs was not carried out. However, an IEP from
Bacillus caldovelox proved to be an excellent candidate for characterisation as
successful purification was achieved. Enzyme engineering was also performed,
fusing a Sac7d domain onto the C-terminus of this protein. These enzymes
were shown to have optimum RT activity at 54ºC with activity still being
displayed at 76ºC. Other studies on these enzymes showed that, unlike the
retroviral RTs, the IEPs displayed no DNA-dependent DNA polymerase activity.
The Sac7d fusion protein was also studied in terms of possible enhancements
to the RT activity of an IEP. However, preliminary studies showed that,
although this domain did not prove to be detrimental to the enzyme, it had little
effect on improving the processivity of the RTs.
thermophilic RT, the IEPs studied in this report did incur major limitations during
cDNA synthesis, which included lower than expected optimum reaction
temperatures, very low fidelity and an inability to synthesise cDNA using
complex RNA templates.
|Date of Award||1 Feb 2011|
|Supervisor||Michael Danson (Supervisor) & David Hough (Supervisor)|
- Reverse transcriptases
- Group II Introns