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BIO 302 - Lecture 09/07/2006

BIO 302 - Lecture 09/07/2006

mRNA Processing

Eukaryotic mRNA processing

Newly made RNA is called “primary transcript” and is modified in three waysbefore leaving the nucleus:

• Cap structure—a modified guanine base is added to the 5’ end.• Poly-A tail—a string of adenine ribonucleotides is added to the 3′ end.• Splicing: Noncoding sequences—called “introns” are removed from the coding sequences,

called the “exons.”

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BIO 302 - Lecture 09/07/2006

RNA Processing

Eukaryotic mRNA processing

• The 5’cap and 3’ tail allow for three other things to occur:

– Facilitate transport out of the nucleus.– Protect the mRNA from degradation.– Promote translation.

BIO 302 - Lecture 09/07/2006

RNA ProcessingEukaryotic mRNA processing

• mRNA Splicing - removal of intron.

Splisosome (Trans acting factors):

– snRNA : small nuclear RNA

– snRNP: small nuclear ribonucloprotein

– Other proteins: Splicing factors

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BIO 302 - Lecture 09/07/2006

RNA Processing

Eukaryotic mRNA processing

Alternative Splicing:

BIO 302 - Lecture 09/07/2006

RNA Processing

Eukaryotic mRNA processing

mRNA Splicing: Chemistry

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BIO 302 - Lecture 09/07/2006

RNA Processing

Eukaryotic mRNA processing

mRNA Splicing: cis acting element

BIO 302 - Lecture 09/07/2006

rRNA Processing

RNA Processing

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BIO 302 - Lecture 09/07/2006

RNA Processing

tRNA ProcessingThe conversion of a yeast tRNA precursor into a mature tRNA requires the removalof a 14-nucleotide intron (yellow), the cleavage of a 5’leader (green), and theremoval of UU and the attachment of CCA at the 3’end (red). In addition, severalbases are modified.

BIO 302 - Lecture 09/07/2006

RNA Processing

tRNA: Aminoacylation

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BIO 302 - Lecture 09/07/2006

RNA Processing

tRNA: Anticodon

Base (#1) in Anticodon Base (#3) in CodonG U or CC GA UU A or GI A, U, or C

mRNA

anticodon loopof tRNA

BIO 302 - Lecture 09/07/2006

Translation

Genetic Code

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BIO 302 - Lecture 09/07/2006

Translation

Genetic Code

BIO 302 - Lecture 09/07/2006

Translation

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BIO 302 - Lecture 09/07/2006

Translation

RibosomeMade of two subunits, large and small.

There are five important sites in the ribosome:(1) mRNA-binding site.(2) A (aminoacyl) site—where tRNA with amino acid enters the ribosome.(3) P (peptidyl) site—new amino acids are attached to the growing amino acid

chain on a tRNA molecule.(4) E (exit) site—tRNAs that do not have an amino acid leave the ribosome (not

shown in the figure below).(5) Catalytic site—forms a peptide bond between two amino acids.

BIO 302 - Lecture 09/07/2006

Translation

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BIO 302 - Lecture 09/07/2006

Translation

Prokaryotic mRNAs:

• Characterized by a Shine-Delgarnosequence that precedes the AUGinitiation codon. Base pairingbetween the Shine-Delgarnosequence and a complementarysequence near the 3´ terminus of16S rRNA aligns the mRNA on theribosome.

Translation Initiation: Signals for initiation (cis acting element)

BIO 302 - Lecture 09/07/2006

Translation

Prokaryotic mRNAs:

• Characterized by a Shine-Delgarnosequence that precedes the AUGinitiation codon. Base pairingbetween the Shine-Delgarnosequence and a complementarysequence near the 3´ terminus of16S rRNA aligns the mRNA on theribosome.

Eukaryotic mRNAs:

• Bound to the 40S ribosomal subunitby their 5´ 7-methylguanosine caps.The ribosome then scans along themRNA until it encounters an AUGinitiation codon.

Translation Initiation: Signals for initiation (cis acting element)

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BIO 302 - Lecture 09/07/2006

Translation Initiation: Signals for initiation (cis acting element)

Translation

BIO 302 - Lecture 09/07/2006

Translation

Initiation

1. The small ribosomal subunit binds to the initiator tRNA.

2. The anticodon of the initiator tRNA with the amino acid base pairs with thecodon AUG of the mRNA to form ribosome subunit–tRNA–methioninecomplex.

3. The ribosome accommodates only one tRNA with a bound amino acidmolecule at the A and P sites of the ribosome.

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BIO 302 - Lecture 09/07/2006

Translation

Elongation

1. Polypeptide chain in the P site is transferred to the new amino acid in the A site.

2. Once the polypeptide chain in the P site has been transferred to the new amino acid in the A site, the Psite has a tRNA without an amino acid.

3. The ribosome moves by one codon on the mRNA. The tRNA not attached to an amino acid leaves theribosome through the E site.

4. The tRNA with the polypeptide (a peptidyl tRNA) is now in the P site.

5. A new codon is in the A site, and a new tRNA with an amino acid enters.

6. Continues as the ribosome shifts and exposes new codons in the 5’ to 3’ direction, and the polypeptideis elongated one amino acid at a time.

BIO 302 - Lecture 09/07/2006

Translation

Elongation

1. Polypeptide chain in the P site is transferred to the new amino acid in the A site.

2. Once the polypeptide chain in the P site has been transferred to the new amino acid in the A site, the Psite has a tRNA without an amino acid.

3. The ribosome moves by one codon on the mRNA. The tRNA not attached to an amino acid leaves theribosome through the E site.

4. The tRNA with the polypeptide (a peptidyl tRNA) is now in the P site.

5. A new codon is in the A site, and a new tRNA with an amino acid enters.

6. Continues as the ribosome shifts and exposes new codons in the 5’ to 3’ direction, and the polypeptideis elongated one amino acid at a time.

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BIO 302 - Lecture 09/07/2006

Translation

Elongation

1. Polypeptide chain in the P site is transferred to the new amino acid in the A site.

2. Once the polypeptide chain in the P site has been transferred to the new amino acid in the A site, the Psite has a tRNA without an amino acid.

3. The ribosome moves by one codon on the mRNA. The tRNA not attached to an amino acid leaves theribosome through the E site.

4. The tRNA with the polypeptide (a peptidyl tRNA) is now in the P site.

5. A new codon is in the A site, and a new tRNA with an amino acid enters.

6. Continues as the ribosome shifts and exposes new codons in the 5’ to 3’ direction, and the polypeptideis elongated one amino acid at a time.

BIO 302 - Lecture 09/07/2006

Translation

Termination

(1) Occurs when the ribosome reaches a stop codon (UAA, UAG, or UGA).

(2) A protein release factor interacts with the stop codon to end translation.

(3) The ribosomal subunits dissociate, all RNAs are released.

(4) The polypeptide is processed or folded, and the protein used by the cell.

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BIO 302 - Lecture 09/07/2006

Post Translational Modification

Examples:

BIO 302 - Lecture 09/07/2006

Control of Gene Expression

(1) Regulates the production of the amino acid tryptophan.

(2) Consists of several elements:– Promoter.– Operator gene overlapping the promoter region.– Five genes encoding enzymes that catalyze the last steps of tryptophan synthesis.

(3) Repressor is inactive unless tryptophan binds to it. The activated repressor attaches to theoperator and blocks transcription.

(4) If there is no tryptophan, the repressor cannot bind and transcription occurs.

(5) Genes are repressed to keep from too much tryptophan production (feedback inhibition)

Trp operon

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BIO 302 - Lecture 09/07/2006

Control of Gene Expression

• Suppose you eliminate trp repressor protein (by deleting the gene encoding for therepressor in the cell), you expect full-length trp mRNA will be synthesized even in thepresence of trp amino acid.

• However, when trp is present in mutant cell: 90 % reduction in level of full length trp mRNA. Accumulation of attenuated RNA [short RNA derived from leader region (L)]

Trp operon

BIO 302 - Lecture 09/07/2006

Control of Gene Expression

Model for attenuation in the trp operon.

a) Proposed secondary structures in E. coli terminated trp leader RNA. Four regions can base pair toform three stem-and-loop structures.

b) When tryptophan is abundant, segment 1 of the trp mRNA is fully translated. Segment 2 enters theribosome (although it is not translated), which enables segments 3 and 4 to base pair. This base-paired region somehow signals RNA polymerase to terminate transcription.

c) In contrast, when tryptophan is scarce, the ribosome is stalled at the codons of segment 1.Segment 2 interacts with segment 3 instead of being drawn into the ribosome, and so segments 3and 4 cannot pair. Consequently, transcription continues.

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BIO 302 - Lecture 09/07/2006

Control of Gene Expression

Model for attenuation in the trp operon.

a) Proposed secondary structures in E. coli terminated trp leader RNA. Four regions can base pair toform three stem-and-loop structures.

b) When tryptophan is abundant, segment 1 of the trp mRNA is fully translated. Segment 2 enters theribosome (although it is not translated), which enables segments 3 and 4 to base pair. This base-paired region somehow signals RNA polymerase to terminate transcription.

c) In contrast, when tryptophan is scarce, the ribosome is stalled at the codons of segment 1.Segment 2 interacts with segment 3 instead of being drawn into the ribosome, and so segments 3and 4 cannot pair. Consequently, transcription continues.

High Trp

Low Trp

BIO 302 - Lecture 09/07/2006

Control of Gene Expression

Lac operon

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BIO 302 - Lecture 09/07/2006

Control of Gene Expression

Lac operon: Promoter sequence

BIO 302 - Lecture 09/07/2006

Control of Gene Expression

Lac operon: Activation by cAMP

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BIO 302 - Lecture 09/07/2006

Control of Gene Expression

Lac operon: Summary

BIO 302 - Lecture 09/07/2006

Control of Gene Expression

a) Eukaryotic cells have more DNA than prokaryotes;also has many noncoding regions.

b) Compartmentalization within the cell Eukaryoticcells contain membrane-bound organelles, whileprokaryotes do not. This requires that proteinsmade in the cytoplasm be transported to organelles.

c) More extensive transcript processing

d) Genes are scattered around the genome and not inoperons, and transcription of all the genes must betightly coordinated.

f) Cell and tissue-specific gene expression – Specificsets of genes are activated and inactivated indifferent cell types, since not every cell needs to useevery gene within the chromosomes.