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FCH 532 Lecture 18
Exam on Friday, Mar. 19
Extra Credit due on Friday
Chapter 31: RNA processing
Messenger RNA splicing
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Pulse-chase labeling studies
indicated two important
characteristics of eukaryotic RNA:
1. Most of the rapidly-synthesized
RNA in the nucleus never reached the
cytoplasm.
2. The rapidly-synthesized nuclear
RNA was much larger on average
than cytoplasmic RNA.
A pulse-chase experiment
Cells
32P-RNA
32P-RNA
+ 32P phosphate for
30 seconds (“pulse”)
+31P for 10
minutes (“chase”)
Heteroduplex analysis-the first
evidence of splicing
Viral DNA
Virus-infected cells
denature
Isolate mRNA
• The
annealing of
viral DNA
also
occurred
Anneal, shadow with heavy metals,
analyze in EM
Heteroduplex analysiscontinued
Expected:
• The appearance of Dloops (displacement
loops) in the DNARNA hybrid indicated
the presence of
regions of DNA that
were transcribed, but
later discarded from
the RNA product.
Intron sequences not present in
mature mRNA
Observed:
Eukaryotic genes: alternating expressed
and unexpressed sequences
• Most eukaryotic genes are intersperesed with unexpressed regions.
• Primary sequences vary greatly in length (~2000 - 20,000 nt); much
larger than expected based on the proteins encoded-heterogeneous
nuclear RNA (hnRNA).
• premRNAs are processed by the excision of internal sequences
(introns) which can be 4-10 times longer in aggregate length than the
expressed seqeuences (exons).
DNA-RNA
heteroduplexes
• Annealing RNA from
virus-infected cells
with viral DNA
revealed the
Interpretation of the EM image:
existence of seven
introns-transcribed
regions of the DNA
removed from the
mature mRNA. How
would you prove the loops were
DNA and not RNA?
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Figure 31-47 The sequence of steps in the production
of mature eukaryotic mRNA as shown for the chicken
ovalbumin gene.
RNA sequence at the exon-intron
junctions
• Introns contain invariant 5’-GU and
AG-3’sequences at their borders
• Internal intron sequences are highly
variable even between closely related
homologous genes.
Exons spliced in two-stage reaction
• 1. The 2’-OH group of a specific intron A residue nucleophilically
attacks the 5’-phosphate at the 5’ intron boundry to create a 2’-5’phosphodiester bond (lariat structure).
• 2. The free 3’-OH group forms a 3’,5’-phosphodiester bond with the 5’
terminal residue of the 3’ exon, thereby splicing the two exons together
and releasing the intron as a larioat with a free 3’-OH.
Overall process of splicing pre-mRNA
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Two trans-esterification reactions
occur: first, between
the G at the 5’ end of
the intron and an A
2’OH near the 3’ end
of the intron; second,
between the
released 3’ end of
the first exon and
and the 5’ end of the
second exon.
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Table 31-4Types of Introns.
Ribozyme activity
• Group I introns found in nuclei, mitochondria,
and chloroplasts. RNA acts as an enzyme in this
group (ribozyme).
• Studies performed by Thomas Cech with
Tetrahymena thermophila.
Figure 31-50 The sequence of
reactions in the self-splicing of
Tetrahymena group I intron.
Page 1260
1. The 3’-OH group of the guanosine
forms a phosphodiester bond with
the intron’s 5’ end, liberating the 5’
exon.
2. The terminal 3’-OH group of 5’
exon forms a phosphodiester bond
with the 5’ terminal phosphate of the
3’ exon-this splices together the two
exons and releases the intron.
3. The 3’-terminal OH group of the
intron forms a phosphodiester bond
with the phosphate of the nucleotide
15 residues form the intron’s 5’ end,
yielding the 5’-terminal fragment with
a cyclized intron.
Ribozyme activity
• Group II introns occur in mitochondria of fungi and plants are also
self-splicing.
• Nuclear pre-mRNA splicing is mediated by spliceosomes.
• Mediated by small nuclear ribonucleoproteins (snRNPs).
• U1-snRNA-U-rich snRNA; partially complementary to the consensus
sequence of the 5’ splice site.
• U2-snRNP, U4-U6-snRNP, U5-snRNP.
U1 and U2 bind to splice
junctions
Splicing mechanism-mRNA transcript
Summary of the three steps in
pre-mRNA processing
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The final mRNA may represent less than 5% of the transcribed
DNA sequence
Alternative splicing and/or poly A sites
increase the effective coding capacity of
eukaryotic genomes
Why introns?
• 1. Evolutionary arguments-represent sites
of recombination between primordial minigenes-the exons
• 2. Function in mRNA export from the
nucleus?
• 3. Allow a variety of protein products from a
single gene by alternative splicing.
• 4. Selfish DNA (no function)
Summary
•RNA processing occurs by a variety of mechanisms to
convert a primary transcript into a final function RNA product
•Eukaryotic pre-mRNAs are capped, polyadenylated, and
spliced to yield one or more mature mRNAs before transport
to the cytoplasm. These processes are coupled in the
nucleus so that only properly processed mRNAs are
exported to the cytoplasm
•The role of introns is still controversial but the favored
hypothesis is that they arose early in evolution and allowed
recombination between mini-genes. They have been almost
eliminated in bacteria and many lower eukaryotes perhaps
because these organisms require a small genome for rapid
replication.
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Translation
Translation-the synthesis of polypeptides
from mRNA.
Genetic code-degenerate codons.
3 nucleotides per codon. Reading frames
can be disrupted by changes in the triplet
codon.
Chemical mutagenesis
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One way of changing the genetic code
occurs through chemical mutagenesisaddition of substances that chemically
induce mutations.
• 2 major classes of mutations:
1. Point mutations-one base pair replaces
another.
a. Transitions-one purine or pyrimidine is
replaced by another.
b. Transversions-purine is replaced by a
pyrimidine or vice versa.
2. Insertion/deletion mutants-one or more
nucleotide pairs are inserted into or
deleted from DNA.
Figure 32-1
Bromouracil.
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•5-bromouracil a base analog that resembles T but because of
the electronegative Br, tautomerizes to to pair with G instead of
A.
•If 5 BU is incorporated into DNA in place of T, can induce A-T
to G-C transitions.
•Can also incorporate in place of C to generate G-C to A-T
transitions.
Page 1286
Figure 32-2 Base pairing by the adenine analog 2aminopurine. It normally base pairs with thymine (a) but
occasionally also does so with cytosine (b).
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Figure 32-3 Oxidative deamination by nitrous acid.
(a) Cytosine ® uracil, which pairs with A.
(b) Adenine ® hypoxanthine, which pairs with C.
Page 1287
Figure 32-4 Reaction with hydroxylamine converts
cytosine to a derivative that base pairs with adenine.
Page 1289
Figure 32-5 The three potential reading frames of an
mRNA. Each reading frame would yield a different
polypeptide.
Genetic code
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mRNAs are read in the 5’  3’ direction.
UAG, UAA, and UGA are Stop codons.
Stop codons are sometimes called
nonsense codons and the individual
codons are called amber (UAG), ochre
(UAA) and opal (UGA) codons.
AUG and GUG are the most typical
start codons - there can be others!
Genetic code
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Genetic code is degenerate-3 amino acids (Arg, Leu,
Ser) are specified by 6 codons.
Most others are specified by 2,3, or 4 codons.
Synonyms - different codons that specify the same
amino acid.
Met and Trp have their own codons (AUG and UGG).
Most synonyms differ only in the third nucleotide position
of the codon.
Most point mutations at a third position in a codon are
silent mutations.
Degeneracy accounts for as much as 33% of the 2575% G+C content among DNAs from different
organisms.
High conc. of Arg, Ala, Gly, and Pro = high G+C content.
High conc. of Asn, Ile, Lys, Met, Phe, and Tyr = low G+C
content.
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Page 1291
Table 32-3 Mitochondrial Deviations from the
“Standard” Genetic Code.
Page 1292
Figure 32-7 The adaptor hypothesis. It postulates that
the genetic code is read by molecules that recognize a
particular codon and carry the corresponding amino
acid.
Page 1293
Figure 32-9 Cloverleaf secondary structure of tRNA.
transfer RNA (tRNA)
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tRNAs have a clover leaf structure.
tRNAs have many modified bases (up to 25%).
5’ phosphate group.
7-bp stem including a 5’ terminal nucleotid that may
contain non-Watson-Crick base pairs (G-U) -acceptor
or amino acid stem.
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The amino acid is attached to the 3’-terminal OH group
to form aminoacyl-tRNA.
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Amino acids are attached by amino-acyl-tRNA
synthetases (aaRSs).
H O O
H O
+ ATP
R-C-C-O-P-O-ribose-adenine
R-C-C
O
+ PPi
NH3+ ONH3+
transfer RNA (tRNA)
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Aminoacyl-AMP + tRNA
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2 classes of aminoacyl-tRNA synthetases
Class I and Class II aaRSs have the same 10 members in
all organisms.
Class I enzymes have two homologous polypeptide
segments not found in other proteins
HIGH and KMSKS
Class II enzymes have 3 other sequences in common with
Class I enzymes.
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aminoacyl-tRNA + AMP
Page 1298
Table 32-4
Characteristics of Bacterial Aminoacyl–
tRNA Synthetases.
transfer RNA (tRNA)
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tRNA anticodons can recognize multiple codons.
Example, yeast tRNAPhe has the anticodon GmAA
Anticodon: 3’-A-A-Gm-5’
Codon:
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5’-U-U-C-3’
3’-A-A-Gm-5’
5’-U-U-U-3’
Non-Watson-Crick base pairing can occur at the 3rd
codon-anticodon position (site of degeneracy).
Wobble hypothesis
assumes that the first two codon-anticodon pairings have
normal Watson-Crick pairing structures.
The third position has some “wobble” that allows for
limited conformational adjustments in pairing geometry.
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Figure 32-25a
Wobble pairing. (a) U · G and I · A
wobble pairs. Both have been observed in X-ray
structures.
Page 1307
Figure 32-25b
Wobble pairing. (b) The geometry
of wobble pairing.
Page 1308
Table 32-5 Allowed Wobble Pairing Combinations in
the Third Codon–Anticodon Position.
Wobble hypothesis
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At least 32 tRNAs are required to translate
all 61 triplet codons. 1 is used for the
initiation codon.
Most cells have > 32 codons.
Mammals have > 150 tRNAs.
Nonsense suppression
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Nonsense mutations (insertion of stop codon
UAG, UAA, UGA) are usually lethal when
inserted into an essential protein and
prematurely terminating translation.
Can be rescued by a second mutation in
another part of the genome (intergenic
suppression).
Nonsense suppressor tRNA add an amino
acid instead of stopping the sequence.
Amber (UAG), ochre (UAA) and opal (UGA)
suppressors have been found.
Suppressors are mutants of minor tRNAs (not all
of the tRNAs in that cell are suppressor tRNAs).
Other suppressors
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Missense suppressors - act similarly to
nonsense suppressors. They substitute one
amino acid for another.
Frameshift suppressors- have 8 nucleotides in
their anticodon loops instead of 7 so the read a
4-base codon.
Page 1309
Table 32-6
Some E. coli Nonsense Suppressors.