Lecture Notes on RNA Structural Elements and Function
Laura Landweber, 9/20/95
Double-stranded helices -------------
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at least 50% DS in solution of any random RNA molecule
incl. mRNA (~55-60% in tRNA, rRNA)
short-range pairings
--> hairpins, loops, bulges, junctions
long-range pairing
intramolecular
structural roles- stabilizing 3D folding
eg: Group I introns, RNase P, rRNA
tested by site-specific mutagenesis
functional roles (stabilization and/or specificity of splicing)
Gp II 5 prime splice site selection mediated by
two exon-binding sites (EBSs)
Gp I 6nt IGS (internal guide sequence) = 5 prime EBS
also stabilized by tert. interactions b/n 2 OHs
intermolecular W.-C. pairing - bimolecular recognition
nuclear pre-mRNA splicing snRNAs
U4-U6: regulatory, pairing disrupted during splicing
U1: recognition, 5 prime splice-site selection
branch point selection, etc.
translation
initiation: Shine-Dalgarno binds 6nt at 3 prime end of 16S rRNA
anticodon-codon interactions
prokaryotic antisense regulation
replication, conjugation, gene expression
specific loop-loop interactions, or
"kissing hairpins" --> complete hybridization of 2 RNAs
RNA editing in trypanosomes - guide RNAs (gRNAs)
more functional roles
protein recognition of ssRNA "presented" by paired regions
--> correct orientation of loop, conformation
(non-sequence-specific protein-RNA interactions incl. phosphate
backbone and protein side chains, eg. Glu-tRNA synthase
protein recognition of duplex regions
DEAD (or DEAH) box proteins, incl pre-mRNA splicing factors
and 2 euk translation IFs
DSRAD unwinds dsRNA in Xenopus and other mammals
A -> I editing protein
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Hairpin Loops |||||||||| |
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non-WC pairing often shortens unpaired regions
(semantic point - not really all "loops")
proof by phylogenetic analysis
eg. 2 tetraloops actually biloops with one non-WC bp closing the loop
wobble GU or unusual GA pair
base stacking also stabilizes "loops"
tetraloops and some 3nt loops like UUU interchangeable in rRNA
=> not interacting w/ proteins
or protein interactions are sequence independent
loops may change conformation on binding, requiring increase in loop
free energy
some more flexible
eg. HIV-1 TAR has many conformations all w/ sim. free energy,
not dominated by one structure, but undergoes conformational
change on binding protein Tat and host proteins
roles in translation initiation, propagation and termination
Shine-Dalgarno (SD) or AUG sequestered in a stem and hairpin loop
--> low rate of translation initiation
(verified by experimental elimination of hairpin by deletion
of nts on one side of the hairpin)
euk hairpins between 5 prime cap and first AUG inhibit translation
hairpins 12-15 nt downstream of AUG enhance init. by stalling 40S
mRNA stability - structures in 3 prime UT
control of translation by binding hairpin upstream of SD in own mRNAs
eg. T4 DNA polymerase
viral coat proteins bind hairpin including AUG of replicase genes
ribosomal frameshifting --> synthesis of 2 or more proteins from one init. site
eg. 5 prime-X, XXY, YYZ-3 prime
can slip into -1 reading frame without disrupting 2/3 bps
facilitated by ribosomal pausing.
Interpretation of UGA as silenocysteine codon instead of stop facilitated
when hairpin immediately downstream of UGA or ribosome stalled
control of iron metabolism by iron-response element (IRE), a stem-hairpin loop
unwinding vs. binding proteins regulate mRNA levels of
ferritin (Fe storage) and transferrin receptor (uptake)
[Fe] high --> incr. ferritin and decr. ferritin receptor synthesis and v.v.
IREs in 5 prime UTR of ferritin mRNA and 3 prime UTR of receptor mRNA
opposite regulation by the same RNA structures!
evolutionary strategy --> concerted response
hairpin binding proteins probably confer stability of mRNAs w/3 prime hairpins
transcription termination signal in proks.
RNA hairpin followed by 6-8 Us (--> unstable dA-rU hybrid --> release)
transcription attenuation in proks by terminator hairpin and a run of Us
at a 5 prime leader of downstream gene inhibits txn of downstream genes
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Mismatches and Internal Loops ------/ \-----
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filled with non-WC base pairs (pairing maximized)
stabilized by tightly bound water molecules
water-mediated H-bonds buttress the structure
effect of widening the major groove
eg. Loop E in euk 5S RNA a 9 nt asymmetric internal loop
actually has 4 non-WC bps (GA, Hoogsteen UA, AA and UU) + a bulged G
experiment: delete a U --> 8 nt loop, less stable (less base pairing)
acts like dsRNA in chemical and enzymatic probing
sens. to RNase V1 and bases less reactive to chemical probes
--> specific protein recognition by TFIIIA
ribosomal proteins also often recognize internal loops in rRNA
HIV-1 Rev protein binds a 10 nt asymmetric internal loop
Gp I intron guanosine binding site = loop between two stems
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Bulge Loops -------+ +------
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cause bends in helices
HIV-1 Tat protein binds TAR element (trans activation response) incr. txn
hairpin with 3 nt bulge, conformation and sequence specific binding
also binds free arg
excess of unpaired As in 16S rRNA often protected from chemical modification
by presumed protein contacts in 30S subunit
nucleophile in Gp II and pre-mRNA 5 prime splice site = a bulged A
Pseudoknots
L3 (spans shallow groove)
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/ \
3 prime <------ +-----\---> 5 prime
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+-----/-------+
\ / L2=0 Classical Pseudoknot with coaxial stacking
\__/ (one turn of the quasi-continuous helix)
L1 (spans deep groove)
any tertiary interaction involving W-C bps b/n 2 regions of unpaired nts
not true topological knots
always defined by two stems (S1 and S2) and 2 or 3 loop regions (appendix 2)
potential for coaxial stacking (appendix 2) by juxtaposition of stems
loop regions can be unstructured
can be long-range interaction
distortion in stacking at junctions of stems and loops
minimum loop sizes can be predicted (and can be really small: L1 can be 1 or 2nt
and span a 5-7 base paired region because of the turn of the helix)
equilibrium between pseudoknot and hairpin conformations
marginally more stable than possible hairpins
conformation labile --> good for control of gene expression
modulated by proteins
offer many sites for interaction with proteins
examples:
rRNA, tRNA, Gp I intron core, etc.
control of translational initiation of E. coli rpsO gene encoding RPS15
tRNA-like structures in translational operators of tRNA synthetase genes