1. Gene Expression: Transcription part 2
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

2. Eukaryotic mRNAs mRNA Production in Eukaryotes
1. Eukaryotic mRNAs have 3 main parts
a. The 5’ leader sequence, or 5’ untranslated region (5’ UTR), varies in length.
b. The coding sequence, which specifies the amino acid sequence of the protein that will be produced during translation. It varies in length according to the size of the protein that it encodes.
c. The trailer sequence, or 3’ untranslated region (3’ UTR) also varies in length and contains information influencing the stability of the mRNA.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

3. General structure of mRNA found in both prokaryotic and eukaryotic cells
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

4. 2. Eukaryotes and prokaryotes produce mRNAs somewhat differently.
Prokaryotes use the RNA transcript as mRNA without modification. Transcription and translation are coupled in the cytoplasm. Messages may be polycistronic.
b. Eukaryotes modify pre-RNA into mRNA by RNA processing. The processed mRNA migrates from nucleus to cytoplasm before translation. Messages are always monocistronic.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

5. Processes for synthesis of functional mRNA in prokaryotes and eukaryotes
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6. Production of Mature mRNA in Eukaryotes
1. Eukaryotic pre-RNAs often have introns (intervening sequences) between the exons (expressed sequences) that are removed during RNA processing. Introns were discovered in 1977 by Richard Roberts, Philip Sharp and Susan Berger.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

7. 5’ and 3’ Modifications: Cap structure at the 5 end of a eukaryotic mRNA
1. The newly made 5’ end of the mRNA is modified by 5’ capping. A capping enzyme adds a guanine, usually 7-methyl guanosine (m7G), to the 5’ end using a 5’-to-5’ linkage. Sugars of the 2 adjacent nt are also methylated. The cap is used for ribosome binding to the mRNA during translation initiation.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

8. 2. The 3’ end of the pre-RNA has 50–250 adenines added enzymatically to form a poly(A) tail. The poly(A) tail is important in mRNA stability, and also plays a role in transcription termination, since RNA polymerase II does not rely directly on a signal in the DNA. Mammals are an example : a. Transcription of mRNA continues through the poly(A) consensus sequence (AAUAAA), the poly(A) site and the GU-rich sequence. b. A protein called CPSF (cleavage and polyadenylation specificity factor) binds the AAUAAA signal. c. A protein called CstF (cleavage stimulation factor) binds to the GU-rich sequence. d. CPSF and CstF bind to each other, producing a loop in the RNA. e. CFI and CFII bind near the poly(A) site, and RNA is cleaved. f. After cleavage, the enzyme poly(A) polymerase (PAP) binds to CPSF and adds A nucleotides to the 3’ end of the RNA, using ATP as a substrate. g. PABII (poly(A) binding protein II) binds the poly(A) tail as it is produced.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

9. Diagram of the 3 end formation of mRNA and the addition of the poly(A) tail
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

10. Introns
1. Removal of introns is necessary for mRNA maturation, as hnRNA (Heteronuclear RNA) becomes functional mRNA.
2. in Philip leder’s lab (1978) it was shown that the mouse β-globin pre-mRNA (part of the cell’s hnRNA) is colinear with the gene that encodes it, while the mature β-globin mRNA is shorter than the gene. The missing RNA was an intron that was removed during RNA processing.
3. Introns are found in most eukaryotic genes that encode proteins, and have also been found in bacteriophage genes.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

11. Processing of Pre-mRNA to Mature mRNA
RNA Splicing
1. Events in eukaryotic mRNA production are summarized in the next Figure. They include:
a. Transcription of the gene by RNA polymerase II.
b. Addition of the 5’ cap.
c. Addition of the poly(A) tail.
d. Splicing to remove introns.
2. RNA splicing requires signals so that the splicing machinery can distinguish between introns and exons. Introns typically begin with 5’-GU, and end with AG-3’, but the splicing signals involve more than just these two small sequences.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

12. General sequence of steps in the formation of eukaryotic mRNA
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

13. 3. Events in splicing together two exons (designated 1 and 2):
a. cleavage occurs at the 5’ splice junction of exon 1 and the intron.
b. The G nucleotide at the free 5’ end of the intron joins with a specific A nucleotide (18-38 nt upstream of the 3’ spice junction) in the branch-point sequence of the intron, forming an RNA lariat structure.
i. in mammals, the branch-point consensus sequence is YNCURAY.
ii. In yeast, the branch point consensus sequence is UACUAAC; its position is more variable than in mammals.
c. The bond forming the lariat is a 2’-5’ phosphodiester linkage between the 5’ phosphate of the free guanine nt at the end of introns, and the 2’ OH of the adenine nt in the branch-point sequence.
d. The introns lariat is excised, and the exons are joined to form a spliced mRNA. The introns RNA is degraded by the cell.
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14. 4. Splicing occurs in the nucleus, mediated by spliceosomes consisting of small nuclear ribonucleoprotein particles (snRNPs) bound to the pre-mRNA. The snRNPs consist of snRNAs associated with proteins.
a. Each of the 6 principal snRNAs (named U1-U6) is associated with proteins to form the snRNPs.
b. Some of the proteins are specific to particular snRNPs, and others are found in all snRNPs.
c. The U4 and U6 snRNAs occur within the same snRNP (U4/U6 snRNP). All the other snRNPs have only a single snRNA.
d. The snRNPs are abundant in the nucleus.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

15. 5. The steps of splicing:
a. U1 snRNP binds the 5’ splice junction of the intron, as a result of base pairing of the U1 snRNA to the intron RNA.
b. U2 snRNP binds the branch-point sequence upstream of the 3’ splice junction.
c. U4/U6 and U5 snRNPs interact, then bind the U1 and U2 snRNPs, creating a loop in the intron.
d. U4 snRNP dissociates from the complex, forming the active spliceosome.
e. The spliceosome cleaves the intron at the 5’ splice junction, freeing it from exon 1. The free 5’ end of the intron bonds to a specific nucleotide (usually A) in the branch-point sequence to form an RNA lariat.
f. The spliceosome cleaves the intron at the 3’ junction, liberating the intron lariat. Exons 1 and 2 are ligated, and the snRNPs are released.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

16. Model for intron removal by the spliceosome
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17. Self-Splicing of Introns in Tetrahymena Pre-rRNA
1. The rRNA genes of most species do not contain introns.
2. Some species of the protozoan Tetrahymena have a 413-bp intron in their 28S rRNA sequence. Tom Cech (1982) showed that splicing of this intron (called a group I intron) is protein- independent. The intron self-splices by folding into a secondary structure that catalyzes its own excision.
3. Other group I introns occur in:
a. The rRNA genes and some mRNA genes in mitochondria of yeast, Neurospora and other fungi.
b. The rRNA genes of all insect species examined.
c. The rRNA and some mRNA and tRNA genes in bacteriophages.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

18. 4. The steps in self-splicing of a group I intron in Tetrahymena:
a. The pre-rRNA is cleaved at the 5’ splice junction and guanosine is added to the 5’ end of the intron.
b. The intron is cleaved at the 3’ splice junction.
c. The two exons are joined together.
d. The excised intron forms a lariat structure, which is cleaved to produce a circular RNA and a short linear piece of RNA.
5. Removal of spacers is a different activity from removal of introns, because spacer removal releases a free rRNA that remains separate, while removal of an intron results in ligation of the RNA sequences that flanked the intron.
6. Self-splicing is not an enzyme activity, because the RNA is not regenerated in its original form at the end of the reaction. However, the discovery of ribozymes (catalytic RNAs) has significantly altered our view of the biochemistry involved in the origin of life.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

19. Self-splicing reaction for the group I intron in Tetrahymena pre-rRNA
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

20. RNA Editing
1. RNA editing adds or deletes nucleotides from a pre-mRNA, or chemically alters the bases, resulting in an mRNA with bases that don’t match its DNA coding sequence. 2. Examples have been found in a number of organisms: a. In Trypanosome brucei (a protozoan causing sleeping sickness) the cytochrome oxidase subunit III gene (from mitochondrial DNA) does not match its mRNA. Uracil residues have been added and removed, and over 50% of the mature mRNA consists of posttranscriptionally added Us. This RNA editing is mediated by a guide RNA (gRNA) that pairs with the mRNA, cleaving it, adding the Us and ligating it. b. In Physarum polycephalum, a slime mold, single C nucleotides are added posttranscriptionally at many positions in mRNAs from several mitochondrial genes. c. In higher plants, many mitochondrial and chloroplast mRNAs undergo C-to-U editing, including production of an AUG initiation codon from an ACG codon in some chloroplast mRNAs. d. In mammals, C-to-U editing occurs in the mRNA for apolipoprotein B, resulting in a tissue-specific stop codon. A-to-G editing occurs in the glutamate receptor mRNA, and pyrimidine editing occurs in some tRNAs.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

21. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 5

22. Transcription of Other Genes: Genes that do not encode proteins are also transcribed, including genes for rRNA, tRNA and snRNA. Ribosomal RNA and Ribosomes: Ribosomes are the catalyst for protein synthesis, facilitating binding of charged tRNAs to the mRNA so that peptide bonds can form. A cell contains thousands of ribosomes.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

23. Ribosome Structure:
1. Ribosomes in both prokaryotes and eukaryotes consist of two subunits of unequal size (large and small), each with at least one rRNA and many ribosomal proteins.
2. E. coli is the model for a prokaryotic ribosome. It is 70S, with 50S and 30S subunits.
a. The 50S subunit contains the 23S rRNA (2,904 nt) and 5S rRNA (120 nt), plus 34 different proteins.
b. The 30S subunit contains the 16S rRNA (1,542 nt), plus 20 different proteins.
3. Eukaryotic ribosomes are larger and more complex than prokaryotic ones, and vary in size and composition among organisms. Mammalian ribosomes are an example; they are 80S, with 60S and 40S subunits.
a. The 60S subunit contains the 28S rRNA (~4,700 nt), the 5.8S rRNA (156 nt) and the 5S rRNA (120 nt), plus about 50 proteins.
b. The 40S subunit contains the 18S rRNA (~1,900 nt) plus about 35 proteins.
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24. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 5

25. rRNA genes and rRNA production in E. coli
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26. Eukaryotes generally have many copies of the rRNA genes. a
Eukaryotes generally have many copies of the rRNA genes. a. The three rRNA genes with homology to prokaryotic rRNA genes are 18S-5.8S-28S, in that order. In the chromosome these genes are tandemly repeated 100–1,000 times to form rDNA repeat units. The 5S rRNA gene copies are located elsewhere in the genome. b. A nucleolus forms around each rDNA repeat unit, and then they fuse to make one nucleolus. Ribosomal subunits are produced in this structure by addition of the 5S rRNA and ribosomal proteins. c. RNA polymerase I transcribes the rDNA repeat units, producing a pre-rRNA molecule containing the 18S, 5.8S and 28S rRNAs, separated by spacer sequences. d. The RNA polymerase I promoter in humans has two domains, a core promoter element overlapping the transcription start site, and an upstream control element (UCE). Two transcription factors bind to the promoter. i. human upstream binding factor (hUBF) bind both promoter elements. ii. SL1 binds the complex of hUBF and DNA. SL1 consists of TBF (TATA binding protein, which is also found in TFIID) and 3 TAFs (TBF- associated factors, fidderent from those in TFIID).
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27. 4. When hUBF and SL1 have bound the promoter, the RNA polymerase I bind and transcription begins. Transcription terminates at specific termination site downstream. 5. Specific cleavage steps free the rRNAs from their transcript as part of pre-rRNA processing that takes place in the complex of pre-rRNA, 5S rRNA and ribosomal proteins. The result is formation of 40S and 60S ribosomal subunits, which are then transported to the cytoplasm.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

28. rRNA genes and rRNA production in eukaryotes
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29. Transcription of Genes by RNA Polymerase III
1. Genes transcribed by RNA polymerase III include: a. The eukaryotic 5S rRNA (120 nt), found in the 60S ribosomal subunit. (This rRNA has no counterpart in the prokaryotic ribosome.) b. The tRNAs (75-90 nt), which occur in repeated copies in the eukaryotic genome. i. Each tRNA has a different sequence. ii. All tRNAs have CCA (added post transcriptionally) at their 3’ ends. iii. Extensive chemical modifications are performed on all tRNAs after transcription. iv. All tRNAs can be shown in a cloverleaf structure, with complementary base pairing between regions to form four stems and loops. Loop II contains the anticodon used to recognize mRNA codons during translation. Folded tRNAs resemble an upside-down “L”. c. Some snRNAs (the others are transcribed by RNA polymerase II).
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30. Cloverleaf structure of yeast alanine tRNA
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31. a. The 5S rDNA has two ICR domains, boxA and boxC.
2. Promoter sequences for 5S rRNA and tRNA genes are typically within the sequences that will be transcribed, hence internal control region (ICR). Promoters for the snRNA genes transcribed by RNA pol III are typically upstream of the genes.
3. Transcription initiation for 5S rRNA and tRNAs requires binding of TFIIIs to the ICR, allowing RNA polymerase III to bind.
a. The 5S rDNA has two ICR domains, boxA and boxC.
b. The tDNA has two ICR domains, boxA and boxB.
4. The ICRs interact with transcription factors TFIIIA, TFIIIB and TFIIIC. Formation of the transcription complex on 5S rDNA
a. TFIIIA is bound to boxC, TFIIIC can bind to boxA.
b. When TFIIIA is bound to boxC, TFIIIC can bind to boxA.
c. TFIIIB then binds to TFIIIA and TFIIIC (not to the DNA directly).
d. TFIIIB functions as a transcription initiation factor by positioning RNA polymerase III correctly on the gene.
e. RNA polymerase III then begins transcription 50 bp upstream from boxA, at the beginning of the gene.
f. Once the transcription factors are positioned on the 5S rDNA, they initiate successive rounds of transcription without dissociating from the DNA.
Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO L 5

32. 5. Transcription termination for the 5S rRNA and tRNA genes uses simple sequences at the 3’ end of the genes.
6. Transcription of 5S rDNA produces a mature 5S rRNA, and no sequences need to be removed.
7. Transcription of tRNA genes produces a pre-tRNA with extra sequences at each end, and introns in the tRNAs for certain amino acids. If present, introns are usually found just 3’ to the anticodon, and in many cases the anticodon pairs with the intron in the pre-tRNA. Introns are removed by a specific endonuclease, and splicing is completed by RNA ligase.
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33. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 5

34. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 5