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Methyltransferases of human +ssRNA viruses
Skořepa, Jan ; Bouřa, Evžen (advisor) ; Otava, Tomáš (referee)
The aim of this bachelor's thesis is a structural and functional description of viral methyltransferases of important human +ssRNA viruses from the families Coronaviridae and Flaviviridae. Over 400 million people are infected with diseases such as Dengue, Yellow fever, or Japanese encephalitis (Flaviviridae) every year. During the current pandemic of the SARS-CoV-2 virus (Coronaviridae), more than 750 million people have already been infected worldwide. Methyltransferases are involced in the synthesiz of the cap structure at the 5' end of the viral RNA, which increases its stability and facilitates translation. A detailed structural understanding of proteins NS5 (Flaviviridae methyltransferase), nsp14 and nsp16 (Coronaviridae methyltransferases) is necessary for the subsequent development of their inhibitors. As antivirals, these could help with the treatment of viral diseases caused by coronaviruses and flaviviruses.
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Effect of promoter sequence on utilization of NAD+ as a substrate for transcription initiation by RNA polymerase
Pinkas, Daniel ; Krásný, Libor (advisor) ; Fišer, Radovan (referee)
For a long time, 5' cap has been thought to be privilege only for eukaryotic organisms in form of 7-methylguanosine cap at the end of mRNA. This was changed only a few years ago. By using methods liquid chromatography and mass spectrometry a new molecule associated with RNA of Escherichia coli has been found. This molecule turned out to be nicotinamide adenine dinucleotide (NAD+ ) attached to 5' end of some small regulatory RNAs (sRNA). Later it has been shown, that RNA polymerase can attach NAD+ at 5' of RNA ab initio, meaning that RNA polymerase can utilize NAD+ as a substrate for transcription initiation. To some extent substrate for transcription initiation is chosen based on promoter sequence. Crucial requirement is presence of adenine at +1 position of DNA coding strand. This thesis focuses on promoter sequence requirements for transcription initiation with NAD+ . As a template for transcription four promoters with different modifications and their chimeras are used: RNA1, Pveg, lac UV5 and rrnB P1. Also, I tried to compare RNA polymerase from E. coli and B. subtilis in terms of transcription initiation substrate usage. Lastly, I describe here isolation of NudC, enzyme that cleaves NAD+ to nicotinamide mononucleotide (NMN) and adenosine monophosphate (AMP). NudC will be used for upcoming...
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