1. Transcription The Relationship Between Genes and Proteins 12 th Week Gihan E-H Gawish, MSc, PhD Ass. Professor Molecular Genetics and Clinical Biochemistry KSU

2. Table of Contents History: linking genes and proteins Getting from gene to protein: transcription  Evidence for mRNA Evidence for mRNA  Overview of transcription Overview of transcription  RNA polymerase RNA polymerase  Stages of Transcription Stages of Transcription Promoter recognition Chain initiation Chain elongation Chain termination  mRNA Synthesis/Processing mRNA Synthesis/Processing  References References

3. History: linking genes and proteins 1900’s Archibald Garrod  Inborn errors of metabolism: inherited human metabolic diseases (more information)more information Genes are the inherited factors Enzymes are the biological molecules that drive metabolic reactions Enzymes are proteins Question: How do the inherited factors, the genes, control the structure and activity of enzymes (proteins)?

4. History: linking genes and proteins Beadle and Tatum (1941) PNAS USA 27, 499–506. Hypothesis:  If genes control structure and activity of metabolic enzymes, then mutations in genes should disrupt production of required nutrients, and that disruption should be heritable. Method:  Isolated ~2,000 strains from single irradiate spores (Neurospora) that grew on rich but not minimal medium. Examples: defects in B1, B6 synthesis. Conclusion:  Genes govern the ability to synthesize amino acids, purines and vitamins.

5. History: linking genes and proteins 1950s: sickle-cell anemia  Glu to Val change in hemoglobin  Sequence of nucleotides in gene determines sequence of amino acids in protein  Single amino acid change can alter the function of the protein Tryptophan synthase gene in E. coli  Mutations resulted in single amino acid change  Order of mutations in gene same as order of affected amino acids

6. From gene to protein: transcription Gene sequence (DNA) recopied or transcribed to RNA sequence Product of transcription is a messenger molecule that delivers the genetic instructions to the protein synthesis machinery: messenger RNA (mRNA)

7. Transcription: evidence for mRNA Brenner, S., Jacob, F. and Meselson, M. (1961) Nature 190, 576–81. Question: How do genes work?  Does each one encode a different type of ribosome which in turn synthesizes a different protein, OR  Are all ribosomes alike, receiving the genetic information to create each different protein via some kind of messenger molecule?

8. Transcription: evidence for mRNA E. coli cells switch from making bacterial proteins to phage proteins when infected with bacteriophage T4. Grow bacteria on medium containing “heavy” nitrogen ( 15 N) and carbon ( 13 C). Infect with phage T4. Immediately transfer to “light” medium containing radioactive uracil.

9. Transcription: evidence for mRNA If genes encode different ribosomes, the newly synthesized phage ribosomes will be “light”. If genes direct new RNA synthesis, the RNA will contain radiolabeled uracil. Results:  Ribosomes from phage-infected cells were “heavy”, banding at the same density on a CsCl gradient as the original ribosomes.  Newly synthesized RNA was associated with the heavy ribosomes.  New RNA hybridized with viral ssDNA, not bacterial ssDNA.

10. Transcription: evidence for mRNA Conclusion  Expression of phage DNA results in new phage-specific RNA molecules (mRNA)  These mRNA molecules are temporarily associated with ribosomes  Ribosomes do not themselves contain the genetic directions for assembling individual proteins

11. Transcription: overview Transcription requires: ribonucleoside 5´ triphosphates:  ATP, GTP, CTP and UTP  bases are adenine, guanine, cytosine and uracil  sugar is ribose (not deoxyribose) DNA-dependent RNA polymerase Template (sense) DNA strand Animation of transcription

12. Transcription: overview Features of transcription: RNA polymerase catalyzes sugar-phosphate bond between 3´-OH of ribose and the 5´-PO 4.RNA polymerase Order of bases in DNA template strand determines order of bases in transcript. Nucleotides are added to the 3´-OH of the growing chain. RNA synthesis does not require a primer.

13. Transcription: overview The three-dimensional structures of RNA polymerases from a prokaryote (Thermus aquaticus) and a eukaryote (Saccharoromyces cerevisiae). The two largest subunits for each structure are shown in dark red and dark blue. The similarity of these structures reveals that these enzymes have the same evolutionary origin and have many mechanistic features in common.

14. Transcription: overview In prokaryotes transcription and translation are coupled. Proteins are synthesized directly from the primary transcript as it is made In eukaryotes transcription and translation are separated. Transcription occurs in the nucleus, and translation occurs in the cytoplasm on ribosomes. Animation

15. comparing eukaryotic and prokaryotic transcription and translation. These two processes are closely coupled in prokaryotes, whereas they are spacially and temporally separate in eukaryotes. (A) In prokaryotes, the primary transcript serves as mRNA and is used immediately as the template for protein synthesis. (B) In eukaryotes, mRNA precursors are processed and spliced in the nucleus before being transported to the cytosol for translation into protein. [After J. Darnell, H. Lodish, and D. Baltimore. Molecular Cell Biology, 2d ed. (Scientific American Books, 1990), p. 230.] Animation

16. Transcription: RNA Polymerase DNA-dependent  DNA template, ribonucleoside 5´ triphosphates, and Mg 2+ Synthesizes RNA in 5´ to 3´ direction E. coli RNA polymerase consists of 5 subunits Eukaryotes have three RNA polymerases  RNA polymerase II is responsible for transcription of protein-coding genes and some snRNA molecules  RNA polymerase II has 12 subunits  Requires accessory proteins (transcription factors)  Does not require a primer

17. Stages of Transcription Promoter Recognition Chain Initiation Chain Elongation Chain Termination Animation

18. Transcription: promoter recognition Transcription factors bind to promoter sequences and recruit RNA polymerase.Transcription factors bind to promoter sequences and recruit RNA polymerase DNA is bound first in a closed complex. Then, RNA polymerase denatures a 12–15 bp segment of the DNA (open complex). The site where the first base is incorporated into the transcription is numbered “+1” and is called the transcription start site. Transcription factors that are required at every promoter site for RNA polymerase interaction are called basal transcription factors.

19. The formation of the active eukaryotic initiation complex. The diagrams represent the complexes formed on the TATA box by the transcription factors and RNA polymerase II. (A) The TFIID complex binds to the TATA box through its TBP subunit. (B) TFIID is stabilized by TFIIA. (C) TFIIB and TFIIH join the complex on the TATA box while TFIIE and TFIIF associate with RNA polymerase II. (D) RNA polymerase is positioned by TFIIB, and its carboxy- terminal domain (CTD) is bound by TFIID. (E) The CTD is phosphorylated by TFIIH and is released by TFIID. The RNA polymerase II is now competent to transcribe mRNA from the gene. The formation of the active eukaryotic initiation complex. The diagrams represent the complexes formed on the TATA box by the transcription factors and RNA polymerase II. (A) The TFIID complex binds to the TATA box through its TBP subunit. (B) TFIID is stabilized by TFIIA. (C) TFIIB and TFIIH join the complex on the TATA box while TFIIE and TFIIF associate with RNA polymerase II. (D) RNA polymerase is positioned by TFIIB, and its carboxy- terminal domain (CTD) is bound by TFIID. (E) The CTD is phosphorylated by TFIIH and is released by TFIID. The RNA polymerase II is now competent to transcribe mRNA from the gene. promoter recognition

20. Promoter recognition: promoter sequences Promoter sequences vary considerably. RNA polymerase binds to different promoters with different strengths; binding strength relates to the level of gene expression There are some common consensus sequences for promoters:consensus sequences  Example: E. coli –35 sequence (found 35 bases 5´ to the start of transcription)  Example: E. coli TATA box (found 10 bases 5´ to the start of transcription)

21. Promoters recognized by E. coli RNA polymerase Promoters recognized by E. coli RNA polymerase

22. Promoter recognition: enhancers Eukaryotic genes may also have enhancers. Enhancers can be located at great distances from the gene they regulate, either 5´ or 3´ of the transcription start, in introns or even on the noncoding strand.located One of the most common ways to identify promoters and enhancers is to use a reporter gene.

23. Promoter recognition: enhancers Modular transcriptional regulatory regions using Pax6 as an activator. (A) Promoter and enhancer of the chick lens δ1 crystallin gene. Pax6 interacts with Sox2 and Maf to activate this gene. (B) Enhancer of the rat somatostatin gene. Pax6 activates this gene by cooperating with the Pdx1 transcription factor. (A after Cvekl and Piatigorsky 1996; B after Andersen et al. 1999.)Cvekl and Piatigorsky 1996 Andersen et al. 1999

24. Promoter recognition: other players Many proteins can regulate gene expression by modulating the strength of interaction between the promoter and RNA polymerase. Some proteins can activate transcription (upregulate gene expression). Some proteins can inhibit transcription by blocking polymerase activity. Some proteins can act both as repressors and activators of transcription.

25. Transcription: chain initiation Chain initiation click-:Chain initiation RNA polymerase locally denatures the DNA. The first base of the new RNA strand is placed complementary to the +1 site. RNA polymerase does not require a primer. The first 8 or 9 bases of the transcript are linked. Transcription factors are released, and the polymerase leaves the promoter region. Figure of bacterial transcription initiation click-.bacterial transcription initiation

26. Stepwise assembly of a transcription-initiation complex from isolated RNA polymerase II (Pol II) and general transcription factors  Once the complete transcription-initiation complex has assembled, separation of the DNA strands at the start site to form an open- complex requires ATP hydrolysis.transcriptionstarthydrolysis  As transcription initiates and the polymerase transcribes away from the promoter, the CTD becomes phosphorylated and the general transcription factors dissociate from the TBP- promoter complex.transcriptionpromoter transcription promoter  Numerous other proteins participate in transcription initiation in vivo.proteins transcriptionin vivo

27. Transcription of DNA into RNA is catalyzed by RNA polymerase, which can initiate the synthesis of strands de novo on DNA templates The nucleotide at the 5′ end of an RNA strand retains all three of its phosphate groups; all subsequent nucleotides release pyrophosphate (PP i ) when added to the chain and retain only their α phosphate (red). The released PP i is subsequently hydrolyzed by pyrophosphatase to P i, driving the equilibrium of the overall reaction toward chain elongation. In most cases, only one DNA strand is transcribed into RNA.nucleotide nucleotides The nucleotide at the 5′ end of an RNA strand retains all three of its phosphate groups; all subsequent nucleotides release pyrophosphate (PP i ) when added to the chain and retain only their α phosphate (red). The released PP i is subsequently hydrolyzed by pyrophosphatase to P i, driving the equilibrium of the overall reaction toward chain elongation. In most cases, only one DNA strand is transcribed into RNA.nucleotide nucleotides

28. Transcription: chain elongation Chain elongation:Chainelongation RNA polymerase moves along the transcribed or template DNA strand. The new RNA molecule (primary transcript) forms a short RNA-DNA hybrid molecule with the DNA template.

29. Transcription: chain termination Most known about bacterial chain terminationbacterial chain termination Termination is signaled by a sequence that can form a hairpin loop. The polymerase and the new RNA molecule are released upon formation of the loop. Review the transcription animation.

30. Transcription: mRNA synthesis/processing Prokaryotes: mRNA transcribed directly from DNA template and used immediately in protein synthesis Eukaryotes: primary transcript must be processed to produce the mRNAprocessed  Noncoding sequences (introns) are removed  Coding sequences (exons) spliced together  5´-methylguanosine cap added  3´-polyadenosine tail added Animation

31. Shortly after RNA polymerase II initiates transcription at the first nucleotide of the first exon of a gene, the 5′ end of the nascent RNA is capped with 7-methylguanylate. Transcription by RNA polymerase II terminates at any one of multiple termination sites downstream from the poly(A) site, which is located at the 3′ end of the final exon. After the primary transcript is cleaved at the poly(A) site, a string of adenine (A) residues is added. The poly(A) tail contains ≈250 A residues in mammals, ≈150 in insects, and ≈100 in yeasts. For short primary transcripts with few introns, polyadenylation, cleavage, and splicing usually follows termination, as shown. For large genes with multiple introns, introns often are spliced out of the nascent RNA before transcription of the gene is complete. Note that the 5′ cap is retained in mature mRNAs.RNA polymerasetranscriptionnucleotide exongene Transcription downstreamexon primary transcriptprimary transcriptsintronsgenesintrons transcriptiongene Shortly after RNA polymerase II initiates transcription at the first nucleotide of the first exon of a gene, the 5′ end of the nascent RNA is capped with 7-methylguanylate. Transcription by RNA polymerase II terminates at any one of multiple termination sites downstream from the poly(A) site, which is located at the 3′ end of the final exon. After the primary transcript is cleaved at the poly(A) site, a string of adenine (A) residues is added. The poly(A) tail contains ≈250 A residues in mammals, ≈150 in insects, and ≈100 in yeasts. For short primary transcripts with few introns, polyadenylation, cleavage, and splicing usually follows termination, as shown. For large genes with multiple introns, introns often are spliced out of the nascent RNA before transcription of the gene is complete. Note that the 5′ cap is retained in mature mRNAs.RNA polymerasetranscriptionnucleotide exongene Transcription downstreamexon primary transcriptprimary transcriptsintronsgenesintrons transcriptiongene Overview of mRNA processing in eukaryotes

32. Transcription: mRNA synthesis/processing Removal of introns and splicing of exons can occur several ways  For introns within a nuclear transcript, a spliceosome is required.spliceosome Splicesomes protein and small nuclear RNA (snRNA) Specificity of splicing comes from the snRNA, some of which contain sequences complementary to the splice junctions between introns and exons  Alternative splicing can produce different forms of a protein from the same gene Alternative splicing  Mutations at the splice sites can cause disease Mutations Thalassemia Breast cancer (BRCA 1)ThalassemiaBreast cancer

33. Transcription: mRNA synthesis/processing RNA splicing inside the nucleus on particles called spliceosomes. Splicesomes are composed of proteins and small RNA molecules (100–200 bp; snRNA). Both proteins and RNA are required, but some suggesting that RNA can catalyze the splicing reaction. Self-splicing in Tetrahymena: the RNA catalyzes its own splicing Catalytic RNA: ribozymes Animation