RNA processing
RNAs in both prokaryotes and eukaryotes are post-transcriptionally modified in some way:
mRNA: splicing, editing, capping, polyadenylation, some chemical modifications
splicing: occurs in both prokaryotes and eukaryotes
genomic DNA sequence contains regions not found in mRNA
genomic sequence transcribed and extra sequences (called introns) spliced out
leaving exons of mature mRNA
most eukaryotic splicing performed by spliceosomal complex:
splice sites determined by sequences at the ends of introns "GTAG rule"
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5 end of intron bound by U1 ribonucleoprotein (commitment complex)
Promotes U2 rnp recognition of internal A residue of intron (complex A)
U4-U5-U6 complex docks with U1-U2 complex (complex B)
Base-pairing interactions between RNAs rearrange:
U6 catalyzes attack by 2OH of internal A to PO4 of 5 end of intron
--> free 5 exon (but held in place by spliceosome) and lariat-shaped intermediate
attack of 3OH of 5 exon at PO4 of 3 exon
--> spliced exons and lariat-shaped intron
reactions are transesterifications:
concerted breaking of one bond while forming a new one
for first step: R = Adenosine2¹, R' = 5'exon, R" = intron+3'exon
for second step: R= 5'exon, R'= intron, R"=3'exon
usually occur in 5 to 3 direction, but some introns not spliced under certain developmental conditions: alternative splicing
some splicing in eukaryotes and prokaryotes doesnt fit rules of spliceosomal splicing. These are the self-splicing group I and group II introns (see below)
capping: 7methylGppp added to 5 end of eukaryotic mRNA during transcription by guanyltransferase
polyadenylation: many eukaryotic mRNAs have polyA tail added during the process of transcription termination. AAUAAA sequence recognized for cleavage. ~250 A residues added by polyA polymerase
Both are important for mRNA stability
editing: trypanosomes- some genomic sequences lack U residues found in mature message. Guide RNAs with polyU tails bind to mRNA, targeting it for editing. The U residues of the tail are spliced in to yield mature mRNA.
modifications: some messages are chemically modified at specific residues
cytosine deamination of apoB mRNA:
truncated form of apolipoproteinB found in intestine
CAA (Gln) codon deaminated to UAA (stop)
rRNA: cleavage, chemical modification
16S, 23S, 5S rRNA plus some tRNAs synthesized in a single transcript
tRNAs cleaved out by RNase P, rest is processed by RNase III, which recognizes stem-loops between the rRNA sequences
modifications many 2´-OMethyl, plus others similar to tRNA
tRNA: cleavage, chemical modification, some introns
cleavage: many tRNAs are transcribed in groups on a single message or interspersed between rRNAs
a variety of exonucleases and endonucleases are responsible for production of mature tRNA:
RNase P: exonuclease cleaves off extra 5 sequences to yield mature tRNA 5 end
3 end from endonuclease cleavage (RNase P or others) followed by a variety of exonucleases. (including RNase D)
In some cases the 3 CCA sequence is added post transcriptionally by tRNA nucleotidyl transferase
mature tRNA
73 - 93 nt in length, most of sequence is variable, but secondary and tertiary structure essentially the same
2° structure - "cloverleaf"
four base-paired helical regions: anticodon stem, D, T and acceptor stems
T and acceptor stem stack into one continuous helix
D and anticodon stack too
4 loops: anticodon, D, T and variable loops. D and T loops interact to form -->
3° structure - elbow shaped
the anticodon loop, which base pairs with mRNA codon is 80Å away from acceptor stem 3 end, where amino acid is attached
3 end always CCA
modifications: a variety of chemical modifications are made to specific bases of tRNA. These help the tRNA fold into proper tertiary structure and aid in the recognition of tRNA by enzymes such as the ribosome
introns: removed by yet another pathway-
cleaved by specific endonuclease and later ligated together
Catalytic RNAs (Ribozymes) |
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Intron splicing |
Satellite RNAs |
RNase P |
Others |
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Group I |
Hammerhead |
Bacterial |
Ribosome! |
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Group II |
Hairpin |
Other species? |
Spliceosome |
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Spliceosome? |
Hepatitis d(HDV) |
Do-it-yourself |
Most involved in making/breaking phosphodiester bonds
All require Mg++ or other divalent cation for activity
Many are associated with proteins in vivo to increase efficiency.
Group I intron - 1st ribozyme discovered
Tetrahymena 26S rRNA gene has ~400nt intron (IVS).
Purified unspliced RNA to study splicing
Added back cell extracts to catalyze reaction,
but reaction occurred in control reaction without adding any proteins!
Protein covalently linked to RNA?
Purified RNA using SDS, phenol, protease digestion, urea gels.
Still worked.
Cloned into E. coli using lac promoter plasmid - transcribed in vitro, purify --> RNA free of Tetrahymena cellular factors.
Still worked.
Intron RNA itself is responsible for self-splicing
Requires Mg++ (mM)and guanosine (uM) co-factors
Reaction occurs under physiological conditions
Mechanism: transesterification, concerted making/breaking of phosphodiester bonds - bond energy conserved.
GOH attacks 3' end of exon1.
for first step: R = guanosine, R' = 5'exon, R" = IVS+3'exon
for second step: R= 5'exon, R'= IVS, R"=3'exon
Isn't acting as a catalyst in vivo, intron is changed during the course of the reaction.
But "L19" (intron final product) is able to perform a variety of catalytic reactions in vitro:
RNA endonuclease
RNA "polymerase" actually a disproportionation
Phosphatase
Turnover 2/min (slow) but k
Group II Intron
Large RNA ( 6 domains)
Domain I binds exon1
Domain V, catalytic
Catalyzes attack of A-2'OH of domain VI at exon1 site.
--> lariat intermediate analogous to that of
spliceosomal splicing.
Homology to spliceosome ==> RNA may be catalytic moiety of spliceosome.
Requires high salt in vitro
Some encode reverse transcriptase necessary for transposition, or protein cofactor to facilitate splicing.
Ribonuclease P (RNaseP)
Cleaves 5'-precursor sequences from tRNA transcripts.
Bacterial enzyme:
Long known to have large RNA and small protein subunits- thought RNA was just playing structural role
recognizes tRNA structure, not sequence
Dissociate subunits: neither active alone in normal buffer (100 mM monovalent, 10 mM Mg++)
But at 10X salt concentration RNA alone is active- protein isn't. RNA binds tRNA as well as holoenzyme
KM kcat
RNA alone 20 nM 0.5/min
holoenzyme 20 nM 10
==> RNA alone has all necessary groups for binding substrate specifically.
Cofactors:
Mg++
Salt to overcome repulsive forces of charged RNAs
Protein required in vivo.
Eukaryotes:
protein required in vitro (10 subunits known in yeast).
perhaps some binding or catalytic functions transferred to protein subunit - or -
protein required for proper folding of RNA.
cannot dissociate and reassociate and retain activity.
Satellite RNA ribozymes:
Viroids - Naked RNAs which infect plants (ASBV-avocado sunblotch)
Virusoid- satellite of plant RNA viruses encapsidated by viral proteins (sTRSV- tobacco ringspot; LSTV-lucerne transient streak).
Both have ssRNA genomes-
can isolate genomic and antigenomic monomers and linear multimers. ==> rolling circle replication.
Found that multimer RNA can self-cleave to form linear monomers with 5'-OH and 2'-3' cyclic phosphate ends.
Linears can self-ligate to form circles.
In vivo conformation not very active, but heat denature, then quick cool --> good activity.
" " slow cool --> low activity.
==> most stable conformation inactive.
Found similar sequence motif around cleavage site (hammerhead).
trimmed away excess sequences --> good activity
Cleaves in presence of low conc. of Mg
(can cleave during transcription)
Connectivity of strands unimportant for reaction.
Can reconfigure into "substrate" strand and "enzyme" strand.
--> true catalyst with few sequence constraints on substrate
smallest natural ribozyme, can use to cleave any sequence , in theory. Heavily studied as possible therapeutic molecule to knock out undesirable mRNA such as retroviruses, oncogenes.
Hairpin
Antigenomic strand of TRSV self-cleaves, but doesn't fit hammerhead motif. Looks more like a hairpin.
Can be dissected into two domains:
Loop A - substrate strand and SBS (substrate binding site)
Loop B - catalytic domain.
Hepatitis
d virusMg and ribozyme active site
Ribozymes coordinate Mg at active site
Mg stabilizes transition state in transesterification or hydrolysis reaction
Why aren't there more ribozymes?
Nucleic acids: information poor (4 possible sidechains, not 20)
info on inside of the molecule, not outside
==> proteins can present greater variety of environments for catalysis
What about DNA?
Some examples of DNA enzymes appearing, but either require Pb++ (more powerful cleaver) or presence of ribose at cleavage site.
Ribose is better leaving group and can both donate and accept protons to aid in catalysis.
Putting the RNA world to work
Take advantage of having active groups and genetic info on same molecule.
In vitro selection or SELEX
Start with Synthesized oligo:
need way to select for molecules you want and get rid of those you don't:
protein binding (aptamers)
column binding " "
self-cleavers or -ligaters (--> change in gel mobility)
Transcribe RNA
Select active molecules
PCR amplify active molecules (reverse transcribe, then PCR)
repeat many cycles
--> collection of molecules selected to do what you want.
Other modern-day aspects of RNA world
Using ribozyme (or aptamers) to cure your favorite disease
Problems:
Delivery to cells
Stability of ribozyme
Accessibility of target
Turnover
The "RNA World"
How did life begin?
RNA is a good candidate for first molecule that could self-replicate
Has genetic info and catalytic functions on same molecule.
Once ribosome available for translation, then could replicate proteins.
Ribosome
Large, highly conserved RNAs plus many proteins.
Cannot isolate pure rRNA and get any activity,
But protease treat ribosome --> some aa-tRNA cleaving activity remains.
But at least one protein still intact. Are proteins necessary for activity or for proper folding?
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Crystal Structure of Ribosome Large (catalytic) subunit Proteins in gold, RNA in gray, active site in green in center. |
Close-up of active site. |
Crystal structure of active site with substrate analog -> no protein near active site!
So the ribosome is a ribozyme. First ribosome was made of RNA, proteins added later.