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UNIT 3. Transcription and Protein Synthesis. Objectives Discuss the flow of information from DNA to RNA to Proteins Explain transcription Differentiate introns and exons State functions of the noncoding regions of mRNA Describe post transcriptional modification of pre-RNA

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  1. UNIT 3 Transcription and Protein Synthesis

  2. Objectives Discuss the flow of information from DNA to RNA to Proteins Explain transcription Differentiate introns and exons State functions of the noncoding regions of mRNA Describe post transcriptional modification of pre-RNA Distinguish between mRNA, tRNA and rRNA Distinguish the roles of ribosomes, tRNA and mRNA in protein synthesis Give detailed description of the process and steps of translation Describe application of SDS-Page gel electrophoresis to protein analysis

  3. DNA to RNA to Proteins

  4. DNA to RNA to Proteins Eukaryotes vs. Prokaryotes In prokaryotes, transcription and translation are coupled • translation begins while the mRNA is still being synthesized.

  5. mRNA in Prokaryotes

  6. DNA to RNA to Proteins Eukaryotes vs. Prokaryotes • In Eukaryotes, transcription and translation are spatially and temporally separated • transcription occurs in the nucleus to produce a pre-mRNA molecule. • pre-mRNA is processed to produce the mature mRNA, which exits the nucleus and is translated in the cytoplasm.

  7. mRNA in Eukaryotes

  8. DNA to RNA to Proteins Eukaryotes vs. Prokaryotes

  9. Transcription • occurs in four main stages: 1) binding of RNA polymerase to DNA at a promoter 2) initiation of transcription on the template DNA strand 3) elongation of the RNA chain 4) termination of transcription along with the release of RNA polymerase and the completed RNA product from the DNA template.

  10. Transcription • Binding of polymerases to the initiation site at the promoter. • Prokaryotic RNA polymerases can recognize the promoter and bind to it directly, but eukaryotic RNA polymerases have to rely on other proteins called transcription factors. • Unwinding of the DNA double helix by helicase.  • Prokaryotic RNA polymerases have the helicase activity, but eukaryotic RNA polymerases do not.  • Unwinding of eukaryotic DNA is carried out by a specific transcription factor. • Synthesis of RNA based on the sequence of the DNA template strand. • RNA polymerases use nucleoside triphosphates (NTPs) to construct a RNA strand. • Termination of synthesis.   • Prokaryotes and eukaryotes use different signals to terminate transcription. 

  11. Promoter • The DNA promoter region in prokaryotes is a stretch of about 40 base pairs adjacent to and including the transcription start point. • The essential features of the promoter are the start point (designated +1 and usually an A), the six-nucleotide -10 sequence, and the six-nucleotide -35 sequence. • The two key sequences are located approximately 10 nucleotides and 35 nucleotides upstream from the start point.

  12. Prokaryotic promoter

  13. Elongation • During elongation, RNA polymerase binds to about 30 base pairs of DNA • At any given time, about 18 base pairs of DNA are unwound, and the most recently synthesized RNA is still hydrogen-bonded to the DNA, forming a short RNA-DNA hybrid (12 bp long, but it may be shorter) • The total length of growing RNA bound to the enzyme and/or DNA is about 25 nucleotides.

  14. Termination • Requires a termination sequence that triggers the end of transcription. • Two classes exist: • rho dependent • a short complementary GC-rich sequence (followed by several U residues) will form a "brake" that will help release the RNA polymerase from the template. • rho independent. • binding of rho to the mRNA releases it from the template.

  15. Termination • rho-dependent – requires a protein called rho to bind to specific sequence.

  16. Termination • rho-independent - depends on a seqence in mRNA which forms a stem loop.

  17. Termination

  18. Transcription in Eukaryotes • Although transcription in eukaryotes is similar to that in prokaryotes, the process is more complex. • Instead of one RNA polymerase, there are three : • RNA polymerase I (localized to the nucleolus) transcribes the rRNA precursor molecules. • RNA polymerase II produces most mRNAs and snRNAs. • RNA polymerase III is responsible for the production of pre-tRNAs, 5SrRNA and other small RNAs. • The mitochondria and chloroplasts have their own RNA polymerases.

  19. Transcription complex • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html#

  20. Transcription in Eukaryotes • Eukaryotic nuclear genes have three classes of promoters which are individual for the three types of RNA polymerases

  21. Transcription in Eukaryotes • Termination signals end the transcription of RNA by RNA polymerase I and RNA polymerase III without the activity of hairpin structures as seen in prokaryotes. • mRNA is cleaved 10 to 35 base-pairs downstream of a AAUAAA sequence (which acts as a poly-A tail addition signal).

  22. RNA processing in Eukaryotes • Ribosomal RNA processing • involves cleavage of multiple rRNAs from a common precursor, with nontranscribed spacers separate the units. • The transcription unit is transcribed by RNA polymerase I into a single long transcript (pre-rRNA)

  23. RNA processing in Eukaryotes • Every tRNA gene is transcribed as a precursor that must be processed into a mature tRNA molecule by: • removal of the leader sequence at the 5΄ end • replacement of two nucleotides at the 3 ΄ end by the sequence CCA (with which all mature tRNA molecules terminate) • chemical modification of certain bases • excision of an intron.

  24. RNA processing in Eukaryotes • Transcription of eukaryotic pre-mRNAs often proceeds beyond the 3΄ end of the mature mRNA. • An AAUAAA sequence located slightly upstream from the proper 3΄ end then signals that the RNA chain should be cleaved about 10-35 nucleotides downstream from the signal site, followed by addition of a poly-A tail catalyzed by poly(A) polymerase.

  25. RNA processing in Eukaryotes • Messenger RNA in eukaryotes • is first made as heterogeneous nuclear mRNA / pre-mRNA then processed into mature mRNA through: • the addition of a 5 prime cap - a guanosine nucleotide methylated at the 7th position • addition of poly-A tails • the splicing out of introns.

  26. Introns and Exons • Eukaryotic genes have interrupted coding sequences. • There are long sequences of bases within the protein-coding sequences of the gene that do not code for amino acids in the final protein product. • The nocoding regions within the gene are called introns (intervening sequences). • The exons (expressed sequences) are part of the protein-coding sequence.

  27. How introns are removed

  28. How introns are removed • The intron loops out as snRNPs (small nuclear ribonucleoprotein particles, complexes of snRNAs and proteins) bind to form the spliceosome. • The intron is excised, and the exons are then spliced together. • The resulting mature mRNA may then exit the nucleus and be translated in the cytoplasm.

  29. Animation –How introns are removed • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html#

  30. Introns and Exons • A typical eukaryotic gene may have multiple exons and introns and the numbers are quite variable. • In many cases the lengths of the introns are much greater than those of the exon sequences. • For instance the ovalbumin gene contains about 7700 base pairs, 1859 of them in exons.

  31. Ovalbumin gene

  32. Functions of Introns • Evidence now exists that introns have many functions, including for regulation and structural purposes, and that many of the roles now hypothesized for introns are plausible but need further elucidation.

  33. Please read at least three of the following articles for tutorial http://www.sciencedaily.com/releases/2009/05/090528203730.htm http://www.sciencedaily.com/releases/2007/07/070712143308.htm http://www.sciencedaily.com/releases/2006/11/061113180029.htm http://www.sciencedaily.com/releases/2009/06/090606105203.htm http://www.sciencedaily.com/releases/2006/04/060404090831.htm http://www.sciencedaily.com/releases/2005/10/051020090946.htm http://www.sciencedaily.com/releases/2009/05/090520140408.htm http://www.sciencedaily.com/releases/2008/11/081104180928.htm http://www.sciencedaily.com/releases/2008/10/081017080145.htm

  34. Roles of ribosomes, mRNA and tRNA in Protein Synthesis

  35. Protein Synthesis • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html#

  36. References/ sources of images • http://evolution.berkeley.edu/evolibrary/article/0_0_0/mutations_03 • usmlemd.wordpress.com/2007/07/14/dna-replication/ • http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/centraldogma.jpg • http://www.usask.ca/biology/rank/demo/replication/cons.rep.gif • http://click4biology.info/c4b/3/images/3.4/SEMICON.gif • http://www.bio.miami.edu/~cmallery/150/gene/sf12x16.jpg • http://publications.nigms.nih.gov/findings/sept08/images/hunt_gene_big.jpg • http://ghr.nlm.nih.gov/handbook/illustrations/duplication.jpg • http://images.google.com.jm/imgres?imgurl=http://ghr.nlm.nih.gov/handbook/illustrations/duplication.jpg&imgrefurl=http://ghr.nlm.nih.gov/handbook/illustrations/duplication&usg=__BgKRLXXos-xRaUqN5EyP7qchszc=&h=400&w=370&sz=38&hl=en&start=2&tbnid=ZfARmmvAKG02xM:&tbnh=124&tbnw=115&prev=/images%3Fq%3Dduplication%2Bmutation%26gbv%3D2%26hl%3Den%26client%3Dfirefox-a%26rls%3Dorg.mozilla:en-US:official%26sa%3DG • http://images.google.com.jm/imgres?imgurl=http://www.phschool.com/science/biology_place/biocoach/images/transcription/euovrvw.gif&imgrefurl=http://www.phschool.com/science/biology_place/biocoach/transcription/tctlpreu.html&usg=__-hoX0ehn3x9zc2nUeciZ8gLYLqQ=&h=288&w=261&sz=20&hl=en&start=10&um=1&tbnid=X11pPXboEKhRIM:&tbnh=115&tbnw=104&prev=/images%3Fq%3Dtranscription%26ndsp%3D18%26hl%3Den%26sa%3DG%26um%3D1 • www.asa3.org/ASA/PSCF/2001/PSCF9-01Bergman.html • http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-21/CB21.html • http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-21/2101.jpg

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