| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
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Readings: Brown, 9: 212-229
Outline:
A. RNA splicing: ... [Brown, Section 9.2.3, 9.3.3]
RNA splicing occurs in the nucleus, on the initial transcripts (pre-mRNA or hnRNA) complexed with the nuclear proteins: hnRNP particles
1. GU-AG rule: Consensus Sequence: Intron-Exon Junction ... [Brown, Fig 9.14]
GU always at 5' end of intron:
Donor site: AG | GUAAGU
AG always at 3' end of intron: Acceptor site: (Py...Py)
12 NCAG
| N
No other requirements ... ANY Donor site can be adjacent to any Acceptor site ... no tissue dependence ... no base pairing or 2· structure requirements bet 5' and 3' ends
2. Common Splicing Mechanisms:
two Transesterification reactions
... [Brown, Fig
9.15]
1. OH- nucleophilic
attack on 5'-P of intron: 1st transesterification reaction
2. Exon A 3'-OH nucleophilic attack on 3'-P of intron: 2nd
transesterification reaction
3. Exons joined; Intron excised, either as linear RNA or as branched
lariat structure
NOTE: Each step is a Transesterification: new phosphodiester bond formed, no water; hence, no energy input is required ...
Four main (1,2,3,4) mechanisms,
in three (A, B, C) general splicing systems:
... [Brown, Table 9.3]
A. Excision of introns by the RNA itself: ribozyme enzyme ... RNA as enzyme ... found in two groups of introns: Group I and Group II
A-1 Group I introns: self-splicing via a 9 stem-loop secondary structure mechanism ... Found in rRNA genes of lower eukaryotes and in fungal mitochondrial genes
A-4 Group II introns: mechanism similar to Spliceosome mechanism, but using intron RNA as catalyst.
B-2. Introns are removed from yeast nuclear tRNA precursors via a process similar to tRNA precursor RNA processing.
C-3. Large complex called Spliceosome recognizes exon-intron sequences to remove introns ... common in nuclear RNAs of higher eukaryotes ... GU-AG introns
B. Eukaryotic mRNA Splicing:
1. Spliceosomes; C-3: snRNPs: ... [Brown, Fig 9.17]
Common eukaryotic nuclear protein-encoding
genes ...
Mechanism of Splicing: Small RNA-Protein particles used
in Spliceosome complex
Used in vitro systems to characterize intermediates ... occur
in Spliceosome in vivo
Spliceosomes are composed of Splicing Factor
proteins (~40) plus small nuclear RNAs (snRNA) found in
nucleoprotein (~10 proteins) complexes ... 50-60 S size
...
These ribonucleoprotein complexes are called snRNP particles, or snurps
The snRNPs are U1, U2, U5,
and U4/U6, containing U1, U2, U4, U5, U6 snRNAs
These are highly conserved among eukaryotes, although yeast species
are larger
Binding of snRNPs to pre-mRNA involves both base-pairing and snRNP
proteins:
snRNP U1:
Structure: 4 stem-loop Domains (A,B,C,D)
5' non-paired end of U1 snRNA: H-bonds to Donor site of pre-mRNA
This pairing is essential for splicing; some pairing sites more
important than others
a. Splicing Reaction: 3 overall Stages:... [Brown, Fig 9.15]
Stage 1: Donor Site is cut,
5' end is covalently attached to a Branch site in the Intron
in a 5'-2' phosphodiester bond forming a Lariat Intermediate
Branch site is found ~30 nucs upstream of Acceptor site; has
Consensus Sequence:
Py 80 N Py 80 Py 87 Pu 75 A 100 Py 95 ... [Brown, Fig 9.14]
Examples: CACUGAC or UACUAAC
Stage 2: Acceptor Site is cut, exons are joined, intron is released as the Lariat
Stage 3: The Lariat is "debranched" and degraded.
b. Role of snRNP particles in the 3 Stages: ... [Brown, Fig 9.17]
1. U1 snRNP binds to Donor site
2. U2 snRNP H-bonds to Branch site of Intron, using ATP hydrolysis and involving a snRNP "auxilliary factor" (U2AF): Commitment to Splicing ... Complex A
3. U1 snRNP interacts with U2 snRNP, bringing Donor site and Branch site together
4. U5/U4/U6 trimer complex now binds to U1 and U2, with ATP hydrolysis; U5 binds the intron Donor Site; U6 binds U2: This is the complete Spliceosome ... Complex B
ATP provides energy in each of these reactions for conformational changes ...
U4 interacts with U6 via H-bonds
5. U5 shifts position from the intron part to the exon part of Donor site, and U2 H-bonds with U6, displacing U4 from its interaction with U6 ... Complex C
1st Transesterification reaction now occurs; enzyme may be the U2-U6 complex
6. U5 snRNP now interacts with the intron Acceptor site, as well as with U2, catalyzing cutting at the intron-exon junction and joining of the two Exons ... 2nd Transesterification Reaction
7. Spliceosome is now disassembled, with release of U1, U2, U4, U5, U6, joined exons, and intron Lariat
8. Intron Lariat is Debranched and degraded
2. Group II Introns; A-4: ... [Brown, Box 9.3]
fungal mitochondrial introns ... fairly uncommon
Similar to nuclear introns:
GU-AG rule; splicing via a Lariat intermediate; the two
transesterification reactions are similar ... [Brown, Fig 9.17]
BUT ... Intron is Self-Splicing, i.e. Intron is the Enzyme,
a Ribozyme
1) Branch site A-OH -> Donor site G; Donor site G-OH -> Acceptor site A
Domains 5 and 6 in RNA 2· structure are similar to U2-U6 and U2-intron interactions; this provides the active site for the RNA catalytic activity
3. Trans-Splicing:
Splicing as seen above is cis-Splicing:
occurs on same RNA molecule
Can "rig" for trans-Splicing, eg via intermolecular
H-bonding
Some trans-Splicing occurs naturally:
SL RNA (spliced leader RNA) in trypanosomes: 35 b leader
RNA
C. tRNA Processing: 5'-
and 3' ends, and Intron Splicing
... [Brown, Fig
9.21, Table 9.5]
tRNA genes are found in Clusters whose expression occurs via a long pre-tRNA; this precursor-tRNA is cleaved and processed to yield mature tRNAs ...
The tRNA within the pre-tRNA assumes the 2 - and 3·-structure of the mature tRNA.
After cleavage events at the 5'- and 3'-ends of pre-tRNA, and splicing out of any introns, several tRNA bases are modified: T, pseudo-U, dihydro-G, inosine, ... [Brown, Table 9.6]
1. Processing at tRNA 5'- and 3'- ends:
5' end processing: Ribonuclease
P (RNaseP):
Ribonucleoprotein, single RNA of 375 bases
RNA moiety catalyzes cleavage in pre-tRNAs, in reactions similar
to the Yeast tRNA splicing reactions, cleaving precisely at 5'-end
of the mature tRNA
In vivo both the RNA and protein components are important; mutations in either moity inactivate the enzyme ...
The RNA catalytic activity may have been the original activity, with proteins first assuming a subsidiary, stabilizing role ... and then later in evolution assuming the catalytic function per se and RNA assuming a more subsidiary role ...
3' end processing:
A combination of endonuclease and exonuclease generate correct
3' end ...
If CCA is absent from 3' end, tRNA nucleotidyl transferase
adds the CCA
2. Yeast tRNA splicing; B-2 cleavage mechanism: ... [Brown, Page 222, Figure]
Separate cleavage and ligation
reactions, rather than short Consensus Sequences and transesterification
Many tRNA genes have single Intron, beginning One Base 3' to the
Anticodon;
Introns are unrelated and no consensus sequences are involved
KEY: Secondary Structure in the tRNA, particularly in the Anticodon Arm, and in the Intron
Experiments using precursor tRNA accumulated in a yeast splicing mutant showed that several reactions are needed:
1. An RNA endonuclease (RNase) cleaves precursor tRNA at both ends of Intron, generating 5'-OH groups and 2',3'-cyclic-P groups !!
2. The two "half" tRNA pieces stay together via H-bonding
3. Pi is added to the 5'-OH of the 3'-exon from GTP by a GTP-kinase
4. The two halves are then ligated together by an RNA ligase that requires ATP in the typical two-step ligase reaction involving a Ligase-AMP covalent intermediate
5. As part of the ligase reaction, the 2',3'-cyclic-P is opened by cyclic phosphodiesterase, yielding 2'-Pi
6. A phosphatase removes the 2'-Pi
These events are summarized in the following Figure
D. rRNA Processing:
1. rRNA genes are also found in clusters in eukaryotes
and prokaryotes (operons)
... [Brown, Fig 9.20; Page 122]
eukaryotes: 45S cleaved via
endonucs mainly -> 18S, 5.8S, 28S rRNA ...
sizes of pre-rRNA vary among eukaryotes
5S rRNA is added from a separate transcription event ...
bacteria: 30S pre-rRNA is cleaved via RNaseIII into -> 16S, tRNA, 23S, 5S, (tRNA)
2. Group I Introns; A-1 splice mechanism: ... [Brown, Fig 9.22]
RNA as Catalysts ... Ribozymes ... [Brown, Table 9.5]
Found in rRNA genes of lower
eukaryotes and in fungal mitochondrial genes
Also found in three genes in E. coli phage T4 !!! ... the only
example of prokaryotic introns ...
Two common properties:
1. Self-Splicing: ability of the RNA to splice itself ...
intramolecular ... RNA catalyst
2. Characteristic Secondary Structure of 9 stem-loops ...
[Brown, Fig 9.23]
a. Tetrahymena rRNA is the best studied of Group I Intron RNA Ribozymes:
Precursor: 35S processed, as
above, into -> 18S, 5.8S, 26S rRNA
26S has one 400 base Intron in some Tetrahymena strains:
This Intron can Self-Splice; requires ions and a G-nuc (GTP, GDP,
GMP, GR)
... [Brown, Fig 9.22]
b. Self-Splicing Reaction ... 3 steps: ... [Brown, Fig 9.22]
1. G 3'-OH attacks 5' end of
Intron
2. 3'-OH of Exon A attacks 5' end of Exon B: exons join
3. Linear Intron, with G(414) at 5' end, now circularizes ...
c. Circularization of the Linear Intron:
1. 3'-OH of Intron, on G(414) , DIFFERENT from the the free G above, attacks pA(16) or pU(20) , yielding cyclized Intron and splitting off 5'end 15 or 19 nucs from the Intron
2. Water hydrolyzes the cyclized Intron at SAME position
3. If First cyclization were at pA(16), then 3-OH of Intron attacks pU(20) , yielding cyclized Intron and splitting of 3 nuc linear piece
4. Water hydrolyzes the cyclized Intron at SAME position, yielding L-19 RNA
L-19 RNA has Enzymatic Activity: cleavage and rejoining of oligonucleotide substrates
d. Role of Intron RNA Secondary Structure, the 9 stem-Loops: ... [Brown, Fig 9.22]
P1 stem (Exon 1 and IGS - Internal Guide Sequence - sequence elements) important in Step 1 of Splicing Reaction
P4 stem (P and Q seq elements) and P7 stem (R and S seq elements) of greatest importance in Step 2 of Splicing Reaction, but also P3, P6, and 2 bases at 3' Intron end
e. These Stems and Ribozyme Catalysis - Binding sites:
Group I introns can catalyze other reactions in vitro ... all such reactions are based on the transesterification reactions of self-splicing and involve the same stem-loop structures, structures which give rise to enzymatic Binding Sites
Self-splicing as viewed from Binding Sites:
1. P1 helix forms Substrate-Binding
Site
2. P7 helix forms G-Binding Site ... occupied by free
G or G(414) ...
This is the Enzyme Active Site
f. L-19 RNA enzymatic activities:
Function:
1) bind RNA substrate to IGS - Substrate binding site
2) Use G(414) or free G in G-binding site
Example: Nucleotidyl transferase
or RNA polymerase
extension of C(5) oligomer to C(6)
oligomer; can repeat to
yield longer oligomers
Other activities: Endoribonuclease;
RNA ligase; Phosphatase
Can mutate the IGS to change the specificity of the Ribozyme
Works with DNA oligodeoxynucleotides, but more slowly and
weakly: "RNA world?"
RNA species as Catalysts: high substrate affinities, low turnover rates
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
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Smith
