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Transcription----Biosynthesis of RNA

Chapter 13. Transcription----Biosynthesis of RNA. Transcription----Biosynthesis of RNA. Introduction. 1. Transcription is the first stage of the process of gene expression.

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Transcription----Biosynthesis of RNA

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  1. Chapter 13 Transcription----Biosynthesis of RNA

  2. Transcription----Biosynthesis of RNA Introduction 1. Transcription is the first stage of the process of gene expression. 2. Transcription processes have to suffer strictly regulation to meet the needof development, morphogenesis and physiological functions of organisms . 3. The products of transcription are RNA

  3. RNA DNA Transcription The process which RNA polymerase catalyzes the yield of RNA (tRNA, mRNA, rRNA ) with one of double strands of DNA as template, NTPs as precursors, in the light of the rule of complementary base pairing.

  4. The requirements of transcription Precursors : NTP (ATP, UTP, GTP, CTP) Template:DNA Enzyme:RNA polymerase, RNA-pol Other proteins

  5. Comparison of replication and transcription Replication Transcription Template both strands of DNA one of DNA strands Precursors dNTP NTP Base pairing A→T, G→C A→U, T→A, G→C Polymerase DNA polymerase RNA polymerase Product DNA tRNA, mRNA, rRNA Primer

  6. General picture of transcription

  7. Transcription----Biosynthesis of RNA The template and enzyme The transcription in prokaryotes The transcription in eukaryotes The processing of post-transcription

  8. Section 1 The template and enzyme

  9. 1. The template : Structural gene: For one gene, only one of DNA strands can serve as template. So, there are template strand ( Watson strand ) and coding strand ( Crick strand ). The most important character of transcription is asymmetric transcription.

  10. DNA Transcription plot 5’……GCAGTACATGTC…………3’ 3’……c g t c a t g t a c a g…………5’ DNA transcription 5’……GCAGUACAUGUC………3’ mRNA translation N…... Ala Val His Val.. ………….C peptide Note：capital letters means the code strand, small letters means the template strand

  11. Asymmetry transcription 5’ 5’ 5’ 3’ 5’ 3’ 5’ Structure gene Template strand Coding strand Arrowhead means the direction of transcription

  12. For asymmetric transcription, there are two meanings: 1) Only one strand of a gene can serve as template, the other which is complimentary to the template strand can’t be transcripted. 2) Not all the template strands of genes are found in the same strand on DNA molecule.

  13. 2. RNA polymerase The enzyme related to transcription is RNA polymerase, which is termed as DNA dependent RNA polymerase (DDRP) or RNA pol, or transcriptase.

  14.          1) RNA polymerase in prokaryotes only one RNA pol has been found: α2ββ’σ(holoenzyme ) α2ββ’( core enzyme ) Holoenzyme Core enzyme

  15. Core enzyme: theRNA polymerase without the  subunit is called core enzyme (2). Holoenzyme:It caninitiate transcription specifically at promoter sites and catalyze polymerization of two free NTPs. Core enzyme: It can notinitiate transcription specifically at promoter sites but can catalyze RNA elongation.

  16. There are more than one kind of  factor in prokaryotes  70carries out the promoter recognition process on their own, mainly responsible for thehousekeeping geneexpression. ‘Housekeeping’ genes are those that encode many proteins needed for routine cell functions and which are therefor expressed at low rates in all cells.  32is responsible for theheat-shock geneexpression under some emergent cases.

  17. RNA polymerase binds to DNA at the Transcriptional startpoint

  18. 2) RNA polymerase in Eukaryotes There arethree kinds of RNA pol: RNA pol I : 45S rRNA RNA pol II :hnRNA RNA polIII :tRNA, 5s rRNA, snRNA

  19. Section 2 RNA synthesis in Prokaryotes Three phases of transcription: Initiation of transcription Elongation of transcription Termination of transcription

  20. 1. Initiation of transcription During initiation, RNA polymerase recognizes promoter site, and then unwinds DNA locally to expose a single-stranded DNA template that can be transcripted. The transcriptional unit in prokaryotes isoperon that consists of two regions on DNA: Regulation region Structural gene region

  21. Operon structure Regulation region Structural gene region I P O 1 2 Inhibitor gene Operator gene Promotor, the region for binding of RNA pol Inhibitive protein substrate -50 -40 -30 -20 -10 1 10 pppG mRNA TATAATPu TTGACA Pribnow box NH2

  22. The steps of transcription initiation 1) The sigma factor in holoenzyme of RNA pol recognizes the promotor and let the whole enzyme to bind with the promotor sequence. 2) To unwind the local region of promotor on DNA. 3) To form a initiation complex of transcription. Holoenzyme- DNA-pppGpN-OH

  23. Transcriptional start site RNA pol +1 -35 3` pppGpN-OH TATAAT TTGACA -10 Transcriptional bubble Holoenzyme of RNA pol pppG-OH + pppN-OH pppGpN-OH + PPi

  24. Two important sequence in prokaryotic promoters: -10 sequence: Located about 10 nucleotides upstream of where transcription will begin. -35 sequence: Located about 35 nucleotides upstream. -35 -10 +1 TATAAT 5` TTGACA Transcriptional start site By convention, the first nucleotide of the template DNA that is transcribed into RNA is denoted +1, the transcriptional start site.

  25. 2. Elongation of transcription The elongation phase of RNA synthesis, which begins after formation of the first bond, is therefore carried out by core enzyme. The transcriptional complex: Core enzyme of RNA pol-DNA template-new RNA

  26. -subunit dissociates from the enzyme, once transcription has been initiated . The Core enzyme(2) moves along the gene, synthesizes a complementary RNA copy to the DNA template, using four ribonucleoside 5` triphosphates (ATP,CTP,GTP,UTP) as precursors. The 3`-OH at the end of the growing RNA chain attacks the  phosphate groups of the incoming ribonucleoside 5` triphosphate to form a 3`5` phosphodiester bond.

  27. Transcription bubble The complex of RNA polymerase, DNA template and new RNA transcript is called a transcription bubble. Because within it there is a region where the DNA double helix has opened up to allow transcription to occur. The RNA transcript forms a transient RNA-DNA hybrid helix with its template strand but then peels away from the DNA as transcription proceeds.

  28. The DNA is unwound ahead of the transcription bubble, and after the transcription complex has passed , the DNA rewind. Direction: RNA is synthesized in the 5`3`direction. DNA template in the 3` 5` direction.

  29. DNA template strand DNA template strand 5` direction 3` OH OH U U OH OH Transcription by RNA polymerase. In each step the incoming ribonucleotide selected is that which can base-pair with the next base of the DNA template strand. In the diagram, the incoming nucleotide is rUTP to base-pair with the A residue of the template DNA, A 3`5`phosphodiester bond is formed, extending the RNA chain by one nucleotide, and pyrophosphate is released. Overall the RNA molecule grows in a 5`3`direction.

  30. Termination of transcription in prokaryotes Transcription continues until a termination signal is reached. There are two forms in the termination: 1) A mechanism of not-dependent on rho () factor 2) A mechanism of dependent on rho () factor

  31. 1）A mechanism of not-dependent on rho () factor The simplest termination signal is a GC-rich region in the template that is a palindrome, followed by an AT-rich sequence. The RNA made from the DNA palindrome is self-complementary and so base-pairs internally to form a hairpin structure followed by a few U residues.

  32. C U G U G G•C C•G C•G G•C C•G 5`---------GCCGCCAGTTCGGCTGGCGGC-------ATTTT-OH 3` 3`---------CGGCGGTCAAGCCGACCGCCG-------TAAAA-OH 5` template Transcription 5`---------GCCGCCAGUUCGGCUGGCGGC-------AUUUU-OH 3` RNA A typical hairpin structure formed by the 3` end of an RNA molecule during termination of transcription. G•C A•U C•G A-U-U-U-U-OH 3` 5`

  33. A mechanism of not-dependent on rho () factor

  34. 2) A mechanism of dependent on rho () factor Those that lack such a structure require an additional protein, called rho (), to allow recognition of the termination site and stop transcription. Rho (), can bind to the 3’ end of RNA and to change the conformation of RNA pol, therefore to loose the binding RNA pol with DNA template, then release out from the bubble.

  35. Promoter Structure gene Termination holoenzyme 5` 1 2 3 4 5 6 7 8 5` 1,2. The gene waiting transcription, the single strand with 3`5` is template strand. 3,4. Initiation ,holoenzyme binds to promoter . 5.the first pppG is added. 6. After  dissociates from the enzyme, the elongation begins. 7. Termination,  factor is added, core enzyme releases; 8. Finish of the transcription. Gppp    5`pppG 5`pppG 5` mRNA Core enzyme

  36. DNA RNA 5 3 RNA pol Ribosome

  37. RNA post-transcriptional modification mRNA: In prokaryotes, mRNA requires little or no modification prior to translation. rRNA: (1) Cleavage: the rRNA precursor molecule is cleaved by specific ribonucleases to yield mature 23s,16s and 5s rRNA. (2) Methylation of some bases and ribose moieties of rRNA also occurs.

  38. 16S rRNA tRNA 23S rRNA 5S rRNA An RNA precursor molecule that is cleaved to yield the 23S, 16S and 5S rRNA and a tRNA molecule; the spacer RNA( open blocks) is degraded during these processing steps.

  39. tRNA: (1)Cleavage: the tRNA precursor molecule is cleaved by specific ribonucleases . (2)Some tRNA molecules further require the addition of the three nucleotides CCA to the 3` end before they can function.

  40. Section 3 RNA synthesis in Eukaryotes

  41. Gene transcription in eukaryotes overview The transcription in eukaryotes and prokaryotes is similar, but also there are some difference. 1) RNA polymerase, there are three kinds of RNA pol in eukaryotes, but only one kind in prokaryotes. 2) The process of transcription are different during the initiation and the termination.

  42. RNA polymerases—pol I, pol II, pol III In eukaryote cells, there are three kinds of RNA polymerases. RNA polymerase I Located in the nucleolus and transcribes the 28s, 18s and 5.8s rRNA genes. RNA polymerase II Located in the nucleoplasm and transcribes mRNA from protein-coding gene as well as most small nuclear RNA(snRNAs) involved in mRNA processing .

  43. RNA polymerase III Located in the nucleoplasm and transcribes the genes fortRNA, 5s rRNA, snRNA, and the 7s RNA associated with the signal recognition particle (SRP) involved in the translocation of proteins across the endoplasmic reticulum membrane.

  44. RNA synthesis in eukaryotes It is similar to the process in prokaryotes. Initiation:RNA polymerase combines with the promoter site upstream of the transcriptional start site. Template: only one strand of a double helix DNA acts as a template. Primer: does not require a primer. Direction: The synthesis direction is 5`→3`.

  45. Transcription of mRNA genes mRNA gene organization Exons The sequence of protein-coding sections in a gene. Introns The sequence of nonprotein-coding sections in a gene.

  46. A B C D Split gene The vast majority of protein-coding genes in eukaryotes are discontinuous. The exons are interrupted by introns. coding region A、B、C、D noncoding region The triplet codons within the exons and the order of exons in the gene is consistent with the amino acid sequence of the encoded polypeptide.

  47. Cis-acting elements in eukaryotes Structural gene Promoter AATAA GC CAAT TATA intron -110bp -25bp exon Modification site Initiation site enhancer -70~-80bp Real termination site (A point for cutting and adding a tail )

  48. Structure and expression of a protein-coding gene in eukaryotes Exon 1 Exon 2 Exon 3 promoter termination region Intron1 Intron2 Primary RNA transcript 5`ppp 5`cap added 3`poly(A) added Cleavage by endonuclease and addition of poly(a) tail 5` AAAA2003` cap poly(a) tail RNA slicing 1 2 3 5` AAAA2003` Transport to cytoplasm via nuclear pore

  49. Initiation of mRNA transcription The enzyme---- RNA polymerase II Promoter Most promoter site include a sequence located about 25 bp upstream (i.e. to the 5` side)of the start site which has the consensus TATAAA and is called the TATA box. TATA box sequence resembles the -10sequence inprokaryotes(TATAAT) except that it is located further upstream.

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