intro_classI - University of Windsor

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Class I (Jan 14, 2002)
Introduction: Ribonucleic acids ( RNAs)
I. Structures
Monomers
(i)
Ribose residue (5-member ring sugar)
2’-OH is the key giving RNAs unique
structures and functions
(ii)
Base residues
a. attach to position 1 of ribose
b. either purine, pyrimidine or their
derivertizes
(iii)
Phosphate (Phosphodiester bonds)
pKa1 ~ 7
pKa2 ~ 6.1
Polymers
(i) Synthesis
- RNA polymerases
DNA Polymerases
need primer
5’  3’ extension
3’  5’ nuclease
RNA Polymerases
no primer
5’  3’ ext
no nuclease
- Chemical synthesis
(ii) Nomenclature
- 5’- and 3’ termini
- Helicies (A form of double-stranded
RNAs)
- Base pairing, and stacking
-Triple basepairs
- Stem and loop structures
Parameters of polynucleotide helices
A Form
Direction of helix rotation
Right
B Form
Right
Z Form
Left
Number of residues
per turn (n)
11
10
12
(6 dimers)
Rotation per residue
(= 360o/n)
33o
36o
-60o
per dimer
~-30o per
residue
Rise in helix per residue (h)
0.255 nm 0.34 nm
0.37 nm
Pitch of helix (=nh)
2.8 nm
3.4 nm
4.5 nm
Accessibility
poor
good
poor
II. Functions of RNAs
2.1 Contemporary functions
RNAs involve in..
a. Transcription
e.g. RNAs are primers for DNA pol
b. Translation
e.g. mRNAs (templates), rRNAs and tRNAs
(assemble of translation machinery).
c. Regulation
e.g. Ribosomal binding sequence, poly A tails, splice
sites, etc.
Contemporary functions: in concert with protein
partners
Ribonucleoprotein
“RNP”
2.2 Prebiotic functions
“When proteins are primitive and RNAs perform
various functions from enzymes to genetic
materials.”
Prebiotic concept
“RNA WORLD”
(1986)
Trigger: Ribozyme discovery
Basic assumptions:
(1)
RNA replication assured genetic continuity
(2)
Basepair interaction had been a key in
replication
(3)
Genetically encoded proteins were not
involved as catalysts
Evidence:
1.
RNAs can perform catalytic
activity. The discovery of two selfcatalytic RNAs, including RNase P and
group I intron. RNAs can catalyze
cleaving and ligation.
2.
3.
4.
DNA synthesis requires
ribonucleotide reductase to convert
ribonucleotides to
deoxyribonucleotides. Ribonucleotide
reductase would probably be the
primitive protein enzyme.
RNA viruses use RNAs as genetic
materials, templates to proteins.
Ancient RNA viruses would have RNA
telomere and RNA-telomerase.
RNA molecules with peptide
backbone or six member-ring sugar
residues (pyranose) are nucleophilic,
stable as templates for base-pairing,
protein synthesis and etc.
Genomic Tag Hypothesis
(Weiner & Maizels, 1987)
tRNA-like molecules can perform various functions
:
a. Replication of ss RNA viruses
b. Replication of duplex DNA plasmids of fungal
mitochondria
c. Replication of retro virus
d. Replication of chromosomal telomeres
Hypothesis is that the tRNA-like structure
evolved as 3’-terminal structures
that tagged ancient RNA genomes
for replication.
Genomic Taq is the “Top half” of tRNA structure
= minihelix
The minihelix would be used as an initiation site for
replication. In other word it is a simple telomere.
Supportive documents found in present biological
systems
1.
RNase P is a ribonucleoprotein enzyme
removing the 5’-leader from the tRNA
precursors.
Why tRNA should be processed?
Possibly RNaseP would have been used to
release functional RNAs from RNA genome.
E. coli ‘s RNase P can function using only
RNA component and process the 3’-terminal
tRNA-like pseudoknot of turnip yellow
mosaic virus.
2.
Nucleotidyltransferase catalyzes the
additing of CCA onto the 3’end of
processed tRNA to ensure the presence
of CCA. This enzyme is highly conserved.
3.
Aminoacylation of tRNA (tRNA
charging) is chemically similar to RNA
polymerization.
4.
Neurospora mitochondrial retroplasmids
require RNA as intermediates.
Similar phenomena are found in..
Is that possible that telomerase could have an
internal genomic tag (CmAn motif)?
Is telomerase a ribonucleoprotein enzyme?
It is true at least in the case of Tetrahymena.
Comparison between telomerase and retroplasmid
Reverase transcriptase.
Telomeraes: universal CmAn motif in eukaryotic
nuclear chromosome ends
3’-CCA RNA motifs  5’ terminal motif
(CmAn, RNA)
(TnGm, DNA)
Possible functions of tRNAs : Templates and
Primers
III. Research tools and techniques
a. RNA isolation
b. In vitro translation reaction
c. Radioactive labelling
d. Reverse transcription
e. Gel electrophoresis
f. Spectrofluorometric methods
(Fluorescense energy transfer)
g. Protection assays
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