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RNA
Known RNAs
t-RNA (transfer). Adaptor in translation
m-RNA (messenger)
r-RNA (ribosomal) 5S RNA, 16S RNA, 23S RNA
RNAi (interfering) - inhibition of gene expression, regulatory
miRNA (micro), siRNA (small interfering)
Srp-RNA (component of Signal Recognition Particle)
Sn-RNA (small nuclear, part of spliceosome)
SnoRNA (small nucleolar)
RNA viruses - viral genomes. Retroviruses (HIV), Coronavirus
(SARS)
RNA functions
• Genomic information storage/transfer
Translation,Transcription, splicing
• Regulatory
RNA processing and editing
• Catalytic (ribozymes RNA-enzyme)
• Structural (?)
mRNA synthesis
The mRNA is formed by adding nucleotide that are complementary
to the template strand
DNA coding strand
DNA
5’
3’
3’
3’
G U C A U U C G G
5’
DNA template strand
5’
RNA
The many faces of RNA
RNA is primarily involved in protein synthesis and comes in
three major types:
Ribosome RNA (rRNA) forms the
skeleton of the ribosome, the
machine which makes proteins
Message RNA (mRNA)
is made by transcription
of DNA and lists the
amino acid sequence of
a protein
Growing
protein
Amino
acid
Transfer RNA
(tRNA) transports
amino acids into
the ribosome
Some nucleic acids contain modified bases. Examples:
Nucleoside bases found in RNA:
O
NH2
N
N
N
N
H
N
HN
adenine (A)
NH
N
N
H
N
H2N
O
NH2
guanine (G)
N
H
O
N
H
cytosine (C)
O
uracil (U)
Examples of modified bases found in tRNA:
O
NH2
CH3
NH2
+
H3C
N
+N
N
N
H
N
HN
H2N
N
N
H
O
+
CH3
N
N
H
NH
HN
O
N
H
O
1-methyladenine (m1A) 7-methylguanine (m7G) 3-methylcytosine (m3C) pseudouracil (Ψ)
An Example: t-RNA
• Double strand
• DNA base pairing:
G-C, A-T
“Canonical”,
“Watson-Crick”
• Single strand
• RNA base pairing: G-C, A-U,
G-U and more
– “Canonical”, “Watson-Crick”
– “GU wobble”
– “AU reverse Hoogsteen”
Single-stranded RNA will fold in on
itself to form all sorts of structures!
DNA vs RNA Structure
DNA
“RNA”
DNA
“RNA”
Hierarchical organization of RNA molecules
Primary structure:
5’
ACCACCUGCUGA 3’
Tertiary structure:
Secondary Structure
Covalent & non-covalent bonds in
RNA
Primary:
Covalent bonds
Secondary/Tertiary
Non-covalent bonds
– H-bonds
–
(base-pairing)
– Base stacking
•
Single-stranded
Primary RNA Structure
A – adenine
C – cytosine
G – guanine
U - uracil
PURINES
PYRIMIDINES
Base Pairing
N
C
G-C, A-U
G-U ("wobble")
& variants
C
N
C
C
C
N
N
C
C
C
cytosine
O
O
C
N
C
C
•
•
•
O
Guanine
N
N
N
C
C
C
C
N
C
N
N
N
C
C
C
O
Uracyl
H donor
N
N
Adenine
acceptor
http://www.imbjena.de/ImgLibDoc/nana/IMAGE_NANA.html#sec_element
RNA: non standard base pairs
Wobble base pairs
Inosine
Uracil
RNA Secondary Structure
C
G
U
A
U
G
RNA Secondary Structure
RNA Structure Representations
Full Description
Circle with chords
E
Mountains
Ordered Tree
Balanced Nested Parenthesis
From Fontana, 2003
Moulton et al.,2002
RNA secondary structure representation
Circular representation:
Bacillus Subtilis RNase P RNA
RNA secondary structures
•
G-C and A-U form hydrogen bonded base pairs and are said to be
complementary
•
Base pairs are approximately coplanar and are almost always stacked
onto other base pairs in an RNA structure. Contiguous base pairs are
called stems.
•
Unlike DNA, RNA is typically produced as a single stranded molecule
which then folds intra-molecularly to form a number of short basepaired stems. This base-paired structure is called RNA secondary
structure.
Secondary structure elements
•Stem Loops (hairpins) at least 4 bases
•Bulge loops
•Interior loops
•Junctions (multiloops)
•Kissing hairpins
•Hairpin-Bulge interactions
•Pseudoknots
Common structural motifs in RNA
Helices
Stem Loops (hairpins) at
least 4 bases
Loops
• Hairpin
• Interior
• Bulge
• Multibranch
Junctions (multiloops)
Pseudoknots
Nested vs. Knotted Structures
3’
L1
S2
S1
5’
3’
:(((::((((:::))))::))):
L2
5’
:((((:[[[))))::]]]:
tRNA H bonds
between distant
regions
Neidle, Stephen. Nucleic Acid Structure and Recognition.
Oxford University Press, 2002, p. 148.
RNA: Hairpins
Single stranded subsequences bounded by base pairs are called loops.
A loop at the end of a stem is called a hairpin loop. Simple substructures
consisting of a single stem and loop are called stem loops, or hairpins.
RNA secondary structures
Single stranded bases within a stem are called a bulge of bulge loop if
the single stranded bases are on only one side of the stem.
If single stranded bases interrupt both sides of a stem, they are called an
internal (interior) loop.
RNA “tertiary interactions”
In addition to secondary structural interactions in RNA, there are also
tertiary interactions, including: (A) pseudoknots, (B) kissing hairpins and
(C) hairpin-bulge contact.
Pseudoknot
Kissing hairpins
Hairpin-bulge
Pseudoknots
Kissing hairpins
tRNA structure
tRNA: small molecules (73 to 93 nucleotides) with cloverleaf secondary structure
RNA can make sharp turns
quickly:
180o in two
nucleotides
RNA Secondary Structure Evolution
From Durbin et al.(1998) Biological Sequence Comparison
δ:
C5’ – C4’ – C3’ – O3’
γ:
O5’ – C5’ – C4’ – C3’
β:
P – O5’ – C5’ – C4’
α:
O3’ – P – O5’ – C5’
ζ:
C3’ – O3’ – P – O5’
ε:
C4’ – C3’ – O3’ – P
Backbone torsion angles
The Ribosome
Ribosome Composition (S = sedimentation coefficient)
Ribosome
Source
E. coli
Whole
Ribosome
70S
Small
Subunit
30S
16S RNA
21 proteins
Rat
cytoplasm
80S
40S
18S RNA
33 proteins
Large
Subunit
50S
23S & 5S
RNAs
31 proteins
60S
28S, 5.8S, &5S
RNAs
49 proteins
Eukaryotic cytoplasmic ribosomes are larger and more
complex than prokaryotic ribosomes. Mitochondrial and
chloroplast ribosomes differ from both examples shown.
5S rRNA
“crown” view
displayed as
ribbons & sticks.
PDB 1FFK
Structures of large & small subunits of bacterial & eukaryotic
ribosomes have been determined, by X-ray crystallography &
by cryo-EM with image reconstruction.
Consistent with predicted base pairing, X-ray crystal
structures indicate that ribosomal RNAs (rRNAs) have
extensive secondary structure.
Putting it all Together –
Major Categories of DNA Binding Proteins
Protein residues that make no
contacts with the DNA are colored
blue, those contacting the sugarphosphate backbone are colored red,
and those making base contacts are
colored yellow. (a) Proteins with a
single binding head: T4
endonuclease V (1vas), PU.1 ETS
domain (1pue). (b) Proteins with a
double binding head: lambda
repressor (1lmb), papillomavirus-1
E2 DNA-binding domain (2bop). (c)
Proteins with an enveloping mode of
binding: NF-kB (1nfk),EcoRI
restriction endonuclease (1eri).
Jones et al. 1999 JMB 287(5) 877
Structure of the E. coli Ribosome
large subunit
tRNA
EF-G
small subunit
mRNA
location
The cutaway view at right shows positions of tRNA (P, E
sites) & mRNA (as orange beads). EF-G will be discussed
later. This figure was provided by Joachim Frank, whose lab
at the Wadsworth Center carried out the cryo-EM and 3D
image reconstruction on which the images are based.
Small Ribosomal Subunit
Š In the translation complex, mRNA threads through a
tunnel in the small ribosomal subunit.
Š tRNA binding sites are in a cleft in the small subunit.
Š The 3' end of the 16S rRNA of the bacterial small
subunit is involved in mRNA binding.
Š The small ribosomal subunit is relatively flexible,
assuming different conformations.
E.g., the 30S subunit of a bacterial ribosome was
found to undergo specific conformational changes
when interacting with a translation initiation factor.
30S ribosomal subunit
PDB 1FJF
Small
ribosomal
subunit of a
thermophilic
bacterium:
rRNA in
monochrome;
proteins in
spacefill display
ribbons
varied colors.
The overall shape of the 30S ribosomal subunit is largely
determined by the rRNA. The rRNA mainly consists of
double helices (stems) connected by single-stranded loops.
The proteins generally have globular domains, as well as
long extensions that interact with rRNA and may stabilize
interactions between RNA helices.
Large ribosome subunit:
The interior of the large
subunit is mostly RNA.
Proteins are distributed
mainly on the surface.
PDB 1FFK
Large
Ribosome
Subunit
Some proteins have long
tails that extend into the
interior of the complex.
These tails, which are
highly basic, interact
with the negatively
charged RNA.
"Crown" view with RNAs blue, in
spacefill; proteins red, as backbone.
The active site domain
for peptide bond
formation is essentially
devoid of protein.
PDB 1FFK
Large
Ribosome
Subunit
Peptidyl transferase is
attributed to 23S rRNA,
making this RNA a
"ribozyme."
A universally conserved
adenosine base serves as
a general acid base
during peptide bond
formation.
"Crown" view with RNAs blue, in
spacefill; proteins red, as backbone.
PDB 1FFK
Protein synthesis takes
place in a cavity within
the ribosome.
Nascent polypeptides
emerge through a tunnel
in the large subunit.
Explore the large
ribosomal subunit.
Large ribosome subunit.
Backbone display with RNAs blue. View
from bottom at tunnel exit.
Literature & www-sites
Eddy, S. Non-coding RNA genes and the modern RNA world.
Nat Rev Genet. 2001 Dec;2(12):919-29. Review.
Eddy, S. “Computational genomics of noncoding RNA genes” Cell. 2002 Apr
19;109(2):137-40. Review.
Fontana (2002) Modelling “evo-devo” with RNA BioEssays 24.12.1164-77
Knudsen, B. and J.J.Hein (2003) "Practical RNA Folding” (In Press, RNA)
Knudsen, B. and J.J.Hein (1999) "Using stochastic context free grammars and molecular
evolution to predict RNA secondary structure (Bioinformatics vol 15.5 15.6.446-454)
Moore (1999) Structural Motifs in RNA Ann.Rev.Biochem. 68.287-300.
Moulton et al. (2000) Metrics on RNA Secondary Structures J.Compu.Biol. 7.1/2.277Perriquet et al.(2003) Finding the common homologous structure shared by two
homologous RNAs. Bioinformatics 19.1.108-116.
http://www.imb-jena.de/RNA.html
http://scor.lbl.gov/index.html
http://www.rnabase.org/metaanalysis/
•
•
•
•
•
•
Useful web sites on RNA
Comparative RNA web site
http://www.rna.icmb.utexas.edu/
RNA world
http://www.imb-jena.de/RNA.html
RNA page by Michael Suker
http://www.bioinfo.rpi.edu/~zukerm/rna/
RNA structure database
http://www.rnabase.org/
http://ndbserver.rutgers.edu/
(nucleic acid database)
http://prion.bchs.uh.edu/bp_type/ (non canonical bases)
RNA structure classification
http://scor.berkeley.edu/
RNA visualisation
http://ndbserver.rutgers.edu/services/download/index.html#rnaview
http://rutchem.rutgers.edu/~xiangjun/3DNA/
THE RIBOSOME
• Two subunits, small and
large.
• Largely RNA with many
small proteins.
• Small subunit: decodes
the mRNA.
• Large subunit: catalyzes
peptide bonds.
THE RIBOSOME
THE RIBOSOME
Small
Subunit
Large
Subunit
THE RIBOSOME
THE RIBOSOME
RIBOSOMAL (RNA) STRUCTURE
RNA = STRUCTURE + ACTIVITY
Note the complex secondary
& tertiary rRNA structure.
LARGE SUBUNIT
RIBOSOMAL (PROTEIN) STRUCTURE
• Proteins [gold] stabilize the RNA structure but allow
conformational changes.
• Proteins [gold] stabilize the RNA structure but allow
conformational changes.
The sites represent transitional
states, not structured pockets.
THE RIBOSOME
MECHANICS OF THE RIBOSOME
1.
2.
3.
4.
The SUBUNITS (rRNA + proteins) are assembled
in the nucleolus, then exported.
The SMALL SUBUNIT binds to the mRNA and
finds the START codon.
A LARGE SUBUNIT attaches to complete the
ribosome and translation begins.
The mRNA is pulled through the ribosome until the
STOP codon is reached.
MECHANICS OF THE RIBOSOME
•
The ribosome has four RNA binding sites:
–
–
The mRNA attaches to one RNA binding site of
the ribosome.
tRNAs occupy three more RNA binding sites of
the ribosome: