Newly made RNA is called primary transcript and is modified in three ways before leaving the nucleus:

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m Eukaryotic mrna processing Newly made RNA is called primary transcript and is modified in three ways before leaving the nucleus: Cap structure a modified guanine base is added to

1 m Eukaryotic mrna processing Newly made RNA is called primary transcript and is modified in three ways before 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. 1

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.

2 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. Eukaryotic mrna processing mrna Splicing - removal of intron. Splisosome (Trans acting factors): snrna : small nuclear RNA snrnp: small nuclear ribonucloprotein Other proteins: Splicing factors 2

3 Eukaryotic mrna processing Alternative Splicing: Eukaryotic mrna processing mrna Splicing: Chemistry 3

4 Eukaryotic mrna processing mrna Splicing: cis acting element r 4

5 t The conversion of a yeast trna precursor into a mature trna requires the removal of a 14-nucleotide intron (yellow), the cleavage of a 5 leader (green), and the removal of UU and the attachment of CCA at the 3 end (red). In addition, several bases are modified. trna: Aminoacylation 5

6 trna: Anticodon anticodon loop of trna mrna Base (#1) in Anticodon G C A U I Base (#3) in Codon U or C G U A or G A, U, or C Genetic Code 6

7 Genetic Code 7

Ribosome Made of two subunits, large and small.

(2) A (aminoacyl) site where trna with amino acid enters the ribosome.

8 Ribosome Made 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. 8

Initiation: Signals for initiation (cis acting element) Prokaryotic mrnas: Characterized by a Shine-Delgarno sequence that precedes the AUG initiation codon.

9 Initiation: Signals for initiation (cis acting element) Prokaryotic mrnas: Characterized by a Shine-Delgarno sequence that precedes the AUG initiation codon. Base pairing between the Shine-Delgarno sequence and a complementary sequence near the 3 terminus of 16S rrna aligns the mrna on the ribosome. Initiation: Signals for initiation (cis acting element) Prokaryotic mrnas: Characterized by a Shine-Delgarno sequence that precedes the AUG initiation codon. Base pairing between the Shine-Delgarno sequence and a complementary sequence near the 3 terminus of 16S rrna aligns the mrna on the ribosome. Eukaryotic mrnas: Bound to the 40S ribosomal subunit by their 5 7-methylguanosine caps. The ribosome then scans along the mrna until it encounters an AUG initiation codon. 9

The anticodon of the initiator trna with the amino acid base pairs with the codon AUG of the mrna

10 Initiation: Signals for initiation (cis acting element) 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 the codon AUG of the mrna to form ribosome subunit trna methionine complex. 3. The ribosome accommodates only one trna with a bound amino acid molecule at the A and P sites of the ribosome. 10

11 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 P site 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 the ribosome 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 polypeptide is elongated one amino acid at a time. 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 P site 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 the ribosome 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 polypeptide is elongated one amino acid at a time. 11

12 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 P site 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 the ribosome 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 polypeptide is elongated one amino acid at a time. 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. 12

Post al Modification Examples: Trp operon (1) Regulates the production of the amino acid tryptophan. (2) Consists of several elements: Promoter. Operator gene overlapping the promoter region.

13 Post al Modification Examples: Trp operon (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 the operator 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) 13

14 Trp operon Suppose you eliminate trp repressor protein (by deleting the gene encoding for the repressor in the cell), you expect full-length trp mrna will be synthesized even in the presence 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)] Model for attenuation in the trp operon. a) Proposed secondary structures in E. coli terminated trp leader RNA. Four regions can base pair to form three stem-and-loop structures. b) When tryptophan is abundant, segment 1 of the trp mrna is fully translated. Segment 2 enters the ribosome (although it is not translated), which enables segments 3 and 4 to base pair. This basepaired 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 3 and 4 cannot pair. Consequently, transcription continues. 14

High Trp Low Trp Model for attenuation in the trp operon. a) Proposed secondary structures in E.

b) When tryptophan is abundant, segment 1 of the trp mrna is fully translated.

This basepaired region somehow signals RNA polymerase to terminate transcription.

15 High Trp Low Trp Model for attenuation in the trp operon. a) Proposed secondary structures in E. coli terminated trp leader RNA. Four regions can base pair to form three stem-and-loop structures. b) When tryptophan is abundant, segment 1 of the trp mrna is fully translated. Segment 2 enters the ribosome (although it is not translated), which enables segments 3 and 4 to base pair. This basepaired 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 3 and 4 cannot pair. Consequently, transcription continues. Lac operon 15

16 Lac operon: Promoter sequence Lac operon: Activation by camp 16

Lac operon: Summary a) Eukaryotic cells have more DNA than prokaryotes; also has

b) Compartmentalization within the cell Eukaryotic cells contain membrane-bound

This requires that proteins made in the cytoplasm be transported to organelles.

and not in operons, and transcription of all the genes must be tightly coordinated.

17 Lac operon: Summary a) Eukaryotic cells have more DNA than prokaryotes; also has many noncoding regions. b) Compartmentalization within the cell Eukaryotic cells contain membrane-bound organelles, while prokaryotes do not. This requires that proteins made in the cytoplasm be transported to organelles. c) More extensive transcript processing d) Genes are scattered around the genome and not in operons, and transcription of all the genes must be tightly coordinated. f) Cell and tissue-specific gene expression Specific sets of genes are activated and inactivated in different cell types, since not every cell needs to use every gene within the chromosomes. 17

GCD3033:Cell Biology. Transcription

GCD3033:Cell Biology. Transcription Transcription Transcription: DNA to RNA A) production of complementary strand of DNA B) RNA types C) transcription start/stop signals D) Initiation of eukaryotic gene expression E) transcription factors