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Transcription

Transcription. transcription. Gene sequence (DNA) recopied or transcribed to RNA sequence. Products of Transcription. 1. Ribosomal RNA (rRNA) - Several rRNAs are vital constituents of ribosomes Transfer RNA (tRNA) - The molecule that

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  1. Transcription

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

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

  4. 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

  5. overview • Features of transcription: • RNA polymerase catalyzes sugar-phosphate bond between 3´-OH of ribose and the 5´-PO4. • 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.

  6. 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.

  7. RNA Polymerase • DNA-dependent • DNA template, ribonucleoside 5´ triphosphates, and Mg2+ • Synthesizes RNA in 5´ to 3´ direction • E. coli RNA polymerase consists of 5 subunits • Sigma factors are a subunit of RNA polymerase. • Sigma factors are needed for promoter binding, but after transcription starts they dissociate.

  8. 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

  9. Stages of Transcription • Promoter Recognition • Chain Initiation • Chain Elongation • Chain Termination

  10. promoter recognition • 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.

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

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

  13. 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: • 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)

  14. “-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

  15. 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. • One of the most common ways to identify promoters and enhancers is to use a reporter gene.

  16. 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.

  17. 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.

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

  19. chain elongation • 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.

  20. chain termination • Most known about bacterial 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.

  21. UUUU RNA 3' end of RNA Termination site

  22. Rho and Termination Rho independenttermination depends on both slowing down the elongation complex, and an AT rich region that destabilizes the elongation complex Rho dependentrequires 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

  23. TerminationRho Independent RNA Pol. RNA Pol. RNA Pol. 5’ RNA RNA 5’ Terminator RNA 5’

  24. TerminationRho Dependent RNA Pol. r r r Help, rho hit me! RNA Pol. RNA Pol. 5’ RNA RNA 5’ Terminator RNA 5’

  25. 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 mRNA • Noncoding sequences (introns) are removed • Coding sequences (exons) spliced together • 5´-methylguanosine cap added • 3´-polyadenosine tail added

  26. mRNA synthesis/processing • Removal of introns and splicing of exons can occur several ways • For introns within a nuclear transcript, a spliceosome is required. • 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 • Mutations at the splice sites can cause disease • Thalassemia • Breast cancer (BRCA 1)

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