1. Testing a base-pairing interaction between the enzyme and substrate in the
Hepatitis Delta Virus (HDV) ribozyme
Mayra Pedraza1
, Kandice Simmons1
, Tai Sung Lee3
, Darrin York3
, Daniel Kellerman2
, and Michael Harris2
Cuyahoga Community College1
, Cleveland, OH
Case Western Reserve University2
, Cleveland, OH
Rutgers University3
, Piscataway, NJ
ABSTRACT
HDV is a small satellite RNA virus that is needed for efficient
replication of the hepatitis viral genome. The HDV ribozyme (HDVrz)
is a small (70 nucleotide) RNA motif that undergoes a self-cleavage
reaction that is essential for HDV replication. Understanding how the
HDVrz catalyzes self-cleavage is important for understanding the
mechanisms of viral pathogenesis. Previous molecular mechanics
simulations of the HDV ribozyme reaction indicated the potential for
formation of a Watson-Crick type base pairing interaction between the
catalytic RNA and its substrate that is necessary in positioning the
nucleophile for catalysis. The modeling implicates that nucleotide A79
in the J2/4 region of the ribozyme pairs with nucleotide U(-1) on the
substrate. To test this hypothesis, we created mutant enzymes and
substrates with all four nucleotide possibilities at each position. We
hypothesized that disruption of base-pairing in this region should
decrease enzyme activity while maintaining the paring should restore
activity. Kinetic results showed that mutation of nucleotide A79 of the
ribozyme has a profound negative effect on catalytic activity.
Preliminary analyses of individual mutants show, however, that some
mutations may be defective due to errors in RNA folding rather than
disruption of base-pairing interactions. Further experimentation will
explore the sensitivity of the U(-1) position to nucleotide analog
substitution in order to gain additional information on its function and
interactions with the ribozyme.
INTRODUCTION
HYPOTHESES
RNA molecules recognize each other by basepairing. The HDV
ribozyme is a catalytic RNA molecule that is needed for efficient
replication of the Hepatitis Virus. Molecular simulations of its active site
indicate the formation of a basepair between the substrate RNA and the
enzyme RNA. We are testing this hypothesis by mutating these two
nucleotides individually and examining the effect in ribozyme activity.
Disruption of the basepair is expected to reduce activity, while restoration
of the basepair should also restore catalytic activity. In order to
accomplish this we require the following-
1. Plasmid DNA to serve as a template for in vitro transcription of the
RNA.
2. RNA synthesized by in vitro transcription.
3. Kinetic analysis of the effects of mutation.
DISCUSSION
As stated in the abstract, we thought that the disruption of base-pairing in
this region should decrease enzyme activity while maintaining the paring
should restore activity. Replications were made to get consistent results
which would have helped us confirm our hypothesis before continuing our
experiment. Unfortunately, we have to reject our hypothesis. The computer
generated picture did not work as we had in mind; the picture showed that
A79 was important for basepairing to the (-1) position, but after continuous
experiment it proved that it was not important. Explain more on the results
itself!!
However, this picture also showed A78 which was located next to A79. Even
though the experiment proved our hypothesis false, we would then replicate
the experiment using A78. Keep future minimal!!
REFERRENCES
Also the references should be listed in the appropriate format,
author(s) first, year of publication, title of publication, title of
journal/book, reference pages.
Cleland, WW, and Cook, PF. (2007) Enzyme Kinetics and Mechanism,
Garland Publishers, London and New York
Lee TS, Giambasu GM, Harris ME, York DM. Characterization of the
Structure and Dynamics of the HDV Ribozyme in Different Stages Along
the Reaction Path. The Journal of Physical Chemistry Letters, 2011,
2:2538-2543
The making of tRNAs and more - RNase P and tRNase Z.
Hartmann RK, Gössringer M, Späth B, Fischer S, Marchfelder A.Prog Mol Biol
Transl Sci. 2009;85:319-68. Review. PMID: 19215776 [PubMed - indexed for
MEDLINE]
Two distinct catalytic strategies in the hepatitis δ virus ribozyme cleavage reaction.
Golden BL. Biochemistry.2011 Nov 8;50(44):9424-33. Epub 2011 Oct 17. Review.
PMID: 22003985 [PubMed - indexed for MEDLINE]
Protein-precursor tRNA contact leads to sequence-specific recognition of 5' leaders
by bacterial ribonuclease P.
Koutmou KS, Zahler NH, Kurz JC, Campbell FE, Harris ME, Fierke CA.J Mol
Biol. 2010 Feb 12;396(1):195-208. Epub 2009 Nov 28.PMID: 19932118
[PubMed - indexed for MEDLINE]
ACKNOWLEDGEMENTS
The project was supported by R25GM049010 from the National Institute of General
Medical Sciences and R01GM096000-2. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the National Institute
of General Medical Sciences, the National Institutes of Health, and National Science
Foundation. I would like to thank Dan Kellerman for guiding me thru this amazing
learning experience and Michael Harris for giving me the opportunity to join his
research team.
For more info please contact:
M.K.PEDRAZA@csuohio.edu
meh2@case.edu
Tri-C Bridges to Success in the Sciences
11000 W. Pleasant Valley Road
Cleveland, Ohio 44130
216-987-5621
METHODS and RESULTS
1. Purify plasmid DNA encoding mutant ribozymes.
2. Synthesize mutant ribozyme RNAs by in vitro transcription.
3. Measuring kinetics for mutant HDV ribozymes and substrates allows us to test effect of chemical structure on
ribozyme functional activity assay.
1.1- Purification of the plasmid DNA from bacteria.
1.2- Nanodrop analysis (UV absorption)
1.3- Linearization of the DNA by restriction enzyme digestion
1.4- Agarose gel analysis of the digestion products
1.5- Extraction and precipitation to prepare the template for in vitro transcription.
1.6- Polyacrylamide gel analysis of the in vitro transcription products
1.7- Extraction and precipitation of the RNA from in vitro transcription
1.8- Kinectics of the RNA using a combinations of different enzyme-substrate
reactions
The UV absorbance of the
solution is proportional to
the DNA concentration.
DNA concentration were
determined using Beer’s
Law.
The polymerase synthesizes the
ribozyme; it uses the double
stranded DNA template to make a
single strand of RNA.
Catalytic RNAs are also known
as ribozymes, have the ability to
catalyze the cleavage of covalent
bonds in RNA strands at specific
sites. We are working on the
Hepatitis delta virus ribozyme in
order to understand how it
catalyzes RNA strand cleavage. In
order to study this process HDV
ribozyme must be made in the lab.
Determination of Plasmid DNA concentration using UV spectrum
Wildtype enzymes with 4 substrate are shown in the three different graphs. The bars are in
comparison to the first bar to the left; it shows the movement of the enzyme-substrate samples. The different
bands are the reactions taken at different time points.