FCH 532 Lecture 18

Exam on Friday, Mar. 19Extra Credit due on FridayChapter 31: RNA processing

Messenger RNA splicing• 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.

Cells + 32P phosphate for30 seconds (“pulse”)

32P-RNA

+31P for 10 minutes (“chase”)

32P-RNA

A pulse-chase experiment

Heteroduplex analysis-the first evidence of splicing

• The annealing of viral DNA also occurred

Viral DNA

denatureVirus-infected cells

Isolate mRNA

Anneal, shadow with heavy metals,analyze in EM

Heteroduplex analysis-continued

• The appearance of D-loops (displacement loops) in the DNA-RNA hybrid indicated the presence of regions of DNA that were transcribed, but later discarded from the RNA product.

Expected:

Observed:

Intron sequences not present inmature mRNA

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 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?

Interpretation of the EM image:

Figure 31-47The sequence of steps in the production of mature eukaryotic mRNA as shown for the chicken

ovalbumin gene.

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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

• Two trans-ester-ification 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.

Table 31-4Types of Introns.

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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 31-50The sequence of reactions in the self-splicing of Tetrahymena group I intron.

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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

• 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 mini-genes-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.

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

• One way of changing the genetic code occurs through chemical mutagenesis-addition 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.P

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1286•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.

Figure 32-2 Base pairing by the adenine analog 2-aminopurine. 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.

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Figure 32-4 Reaction with hydroxylamine converts cytosine to a derivative that base pairs with adenine.

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Figure 32-5 The three potential reading frames of an mRNA. Each reading frame would yield a different

polypeptide.

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Genetic code

• 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• 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 25-

75% 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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Table 32-3 Mitochondrial Deviations from the “Standard” Genetic Code.

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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.

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Figure 32-9 Cloverleaf secondary structure of tRNA.P

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transfer RNA (tRNA)• 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.

• The amino acid is attached to the 3’-terminal OH group to form aminoacyl-tRNA.

• Amino acids are attached by amino-acyl-tRNA synthetases (aaRSs).

R-C-C

H

O-

NH3+

O+ ATP R-C-C-O-P-O-ribose-adenine

H

NH3+

O

+ PPi

O

O-

transfer RNA (tRNA)• Aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP

• 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.

Table 32-4 Characteristics of Bacterial Aminoacyl–tRNA Synthetases.

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transfer RNA (tRNA)

• 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.

3’-A-A-Gm-5’

5’-U-U-C-3’

3’-A-A-Gm-5’

5’-U-U-U-3’

Anticodon:

Codon:

• tRNA anticodons can recognize multiple codons.• Example, yeast tRNAPhe has the anticodon GmAA

Figure 32-25a Wobble pairing. (a) U · G and I · A wobble pairs. Both have been observed in X-ray

structures.

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Figure 32-25b Wobble pairing. (b) The geometry of wobble pairing.

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Table 32-5 Allowed Wobble Pairing Combinations in the Third Codon–Anticodon Position.

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Wobble hypothesis

• 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

• 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

• 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.

Table 32-6 Some E. coli Nonsense Suppressors.

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