2. Transcription

3. transcription Gene sequence (DNA) recopied or transcribed to RNA sequence Gene sequence (DNA) recopied or transcribed to RNA sequence

4. Products of Transcription 1. Ribosomal RNA (rRNA) - Several rRNAs are vital constituents of ribosomes vital constituents of ribosomes 2 Transfer RNA (tRNA) - The molecule that physically couples nucleic acid codons with specific amino acids physically couples nucleic acid codons with specific amino acids 3 Messenger RNA (mRNA) - The nucleic acid messenger that carries encoded information from genes on DNA to the protein manufacturing ribosomes messenger that carries encoded information from genes on DNA to the protein manufacturing ribosomes

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

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

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

8. RNA Polymerase DNA-dependent DNA-dependent DNA template, ribonucleoside 5´ triphosphates, and Mg 2+ DNA template, ribonucleoside 5´ triphosphates, and Mg 2+ Synthesizes RNA in 5´ to 3´ direction Synthesizes RNA in 5´ to 3´ direction E. coli RNA polymerase consists of 5 subunits E. coli RNA polymerase consists of 5 subunits Sigma factors are a subunit of RNA polymerase. Sigma factors are a subunit of RNA polymerase. Sigma factors are needed for promoter Sigma factors are needed for promoter binding, but after transcription starts they binding, but after transcription starts they dissociate. dissociate.

9. Eukaryotes have three RNA polymerases Eukaryotes have three RNA polymerases RNA polymerase II is responsible for transcription of protein-coding genes and some snRNA molecules RNA polymerase II is responsible for transcription of protein-coding genes and some snRNA molecules RNA polymerase II has 12 subunits RNA polymerase II has 12 subunits Requires accessory proteins (transcription factors) Requires accessory proteins (transcription factors) Does not require a primer Does not require a primer

10. Stages of Transcription Promoter Recognition Promoter Recognition Promoter Recognition Promoter Recognition Chain Initiation Chain Initiation Chain Initiation Chain Initiation Chain Elongation Chain Elongation Chain Elongation Chain Elongation Chain Termination Chain Termination Chain Termination Chain Termination

11. promoter recognition Transcription factors bind to promoter sequences and recruit RNA polymerase. Transcription factors bind to promoter sequences and recruit RNA polymerase. 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). 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. The site where the first base is incorporated into the transcription is numbered “+1” and is called the transcription start site.

12. Defined regions are transcribed upstream region transcribed region downstream region promoter (RNA polymerase binding site) transcription start site termination site gene dsDNA TB

13. Transcription factors that are required at every promoter site for RNA polymerase interaction are called basal transcription factors. Transcription factors that are required at every promoter site for RNA polymerase interaction are called basal transcription factors.

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

15. “ -35 squence” “Pribnow box (-10 Sequence) Ptrp TTGACA----17bp----TTAACTA---transcription Plac uv5 TTTACA----18bp----TATAATG---transcription Ptac TTGACA----16bp---TATAATG---transcription Prokaryotic TTGACA TATAAT consensus Human ß-globin CCAAT-----39bp----CATAAA----transcription Eukaryotic CCAAT ATA consensus Sekuen DNA dan beberapa promotor bakteri

16. enhancers Eukaryotic genes may also have 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. 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. One of the most common ways to identify promoters and enhancers is to use a reporter gene.

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

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

19. RNA Pol.Initiation T. F. RNA Pol. 5’ RNA Promoter T. F.

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

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

22. UUUU RNA 3' end of RNA Termination site

23. Rho and Termination Rho independent termination depends on both slowing down the elongation complex, and an AT rich region that destabilizes the elongation complex Rho independent termination depends on both slowing down the elongation complex, and an AT rich region that destabilizes the elongation complex Rho dependent requires a protein called Rho, that binds to and slides along the RNA transcript. The terminator sequence slows down the elongation complex, Rho catches up and knocks it off the DNA Rho dependent requires a protein called Rho, that binds to and slides along the RNA transcript. The terminator sequence slows down the elongation complex, Rho catches up and knocks it off the DNA

24. RNA Pol. 5’ RNA Pol. 5’ RNA Pol. 5’ RNA Termination Rho Independent Terminator

25. RNA Pol. 5’ RNA Termination Rho Dependent Terminator  RNA Pol. 5’ RNA  Pol. 5’ RNA Help, rho hit me! 

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

28. mRNA synthesis/processing Removal of introns and splicing of exons can occur several ways Removal of introns and splicing of exons can occur several ways For introns within a nuclear transcript, a spliceosome is required. For introns within a nuclear transcript, a spliceosome is required.spliceosome Splicesomes protein and small nuclear RNA (snRNA) 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 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 can produce different forms of a protein from the same gene Alternative splicing Alternative splicing Mutations at the splice sites can cause disease Mutations at the splice sites can cause disease Mutations Thalassemia Breast cancer (BRCA 1) Thalassemia Breast cancer (BRCA 1) ThalassemiaBreast cancer ThalassemiaBreast cancer