RNA biosynthesis

RNA的生物合成（转录）英文版，主要分为Template and enzyme of transcription in prokaryotes和The process of transcription in eukaryotes两部分。

RNA biosynthesis

• Template and enzyme of transcription in prokaryotes
  • The template of transcription
    • one of the two DNA strands in those segments
      • coding strand
      • template strand
      • Asymmetric transcription
        • only one of the two DNA strands serves as a template.
        • The template strand of different genes is not always on the same strand of DNA.
  • transcription orientation
    • 5'---3'
  • The enzyme:RNApol
    • ·Highest activity after binding dsDNA
    • Structure
      • Holoenzyme：2α，β，β’，w，σ
        • Holoenzyme catalyzes transcription initiation
        • σ：Identify the starting point
      • Core enzyme:2α，β，β’，w
        • Core enzyme catalyzes transcription elongation
  • chemical reaction
  • No need primer
  • Operon
    • consists of clustered numerous structural genes and upstream regulatory sequences. It is prokaryotic transcription units
  • promoter
    • binding sites of RNA pol and DNA template to initiate transcription
    • How to determine the sequence of the promoter?
    • RNA polymerase protection method
    • Promoter in prokaryotes
      • -35region:TTGACA
      • -10region:TATAAT(pribnow box)
      • function:Promoter determines transcription direction and basic transcription level.
  • The transcription process
    • initiation
      • step
        • Step 1: A closed transcription complex is formed.
        • Step 2: An open transcription complex forms in succession.
        • Step3: the formation of the first 3’,5’- phosphodiester bond
      • points
        • Holoenzyme required
        • Primer not required
        • First nucleotide often is GTP/ATP: 5’-pppGpN-OH3’
    • Eelongation
      • 1.Promoetr clearance
        • Abortive initiation is overcome
        • The σ subunit dissociates as the first ~10nt of new RNA are synthesized. (holoenzyme → core enzyme)
        • RNA pol leaves the promoter to elongate RNA
      • 2.Transcription bubble
        • RNA pol generally keeps ~17 bp DNA unwound
        • The DNA/RNA heteroduplex is short (~8bp) and transient.
        • G≡C > A=T > A=U
      • features
        • Core enzyme
        • A=U
        • 5’→3’ elongation
        • Form transcription bubble (Dynamic 8 bp RNA-DNA hybrid)
        • Coupled transcription and translation
      • Coupled transcription and translation in prokaryotes
        •  one gene usually is transcribed by many molecules of RNA polymerasesimultaneously.
        • Each mRNA is being translated by many ribosomes simultaneously
    • Termination
      • ρ-dependent termination
        • Rho factor(ρ）
          • RNA binding protein.
          • Rho factor consists of 6 units, each weighs 46kD.
          • High affinity for transcript RNA especially for polyC.
          • ATPase: hydrolyzes ATP
          • Helicase: disrupts the RNA-DNA hybrid by hydrolysis of ATP
        • Features
          • The ρ-dependent terminators usually include a CA-rich sequence called a rut (rho utilization) element.
          • rut sequence locates at 3’ end of RNA. 
          • The ρ protein associates with the RNA at rut site.
        • The processs
          • The ρ helicase binds to a rut site
          • The ρ helicase migrates along the mRNA to the elongatingRNA polymerase
          • The ρ helicase separates the mRNA from the DNA temple
      • ρ-independent termination
        • Features
          • a region producing an RNA transcript with self-complementary sequences, permitting the formation of a hairpin structure near 3’ end of the RNA strand.
          • a highly conserved string of A residues in the template strand that are transcribed into string of U residues near the 3’ end of the hairpin
        • The process
          • Hairpin disrupts part of the RNA-DNA hybrid.
          • Hybrid duplex contains a particularly unstable base pairing (A-U)
• The process of transcription in eukaryotes
  • Eukaryotic RNA pols
    • RNA pol Ⅱ
      • 12 subunits
      • The largest RPB1 subunit has C-terminal domain
      • CTD
        • a long tail consisting of many repeats of a consensus AAs: Tyr-Ser-Pro-Thr-Ser-Pro-Ser
        • Unphosphorylated CTD:---initiation
        • Phosphorylated CTD ---elongation
    • RNA pol Ⅱ requires an array of general TFs to form the active transcription complex
  • Cis-acting element
    • Core promoter: TATA box (Hognest box), -30~-25bp
    • Upstream element: CAAT box, GC box, -200~-70bp
    • Distant regulatory sequence: enhancer, silencer
  • Transcription factor (TF)
    • Trans-acting factor: recognizes and binds with cis-acting element directly or indirectly
    • TF classification
      • general TF:
        • binds to RNA-pol directly or indirectly
      • specific TF:
        • mediator, upstream factor
      • TF name
        • TFI, II, III for RNA pol I, II, III
    • function of TFⅡ
      • TBP
        • Specifically recognizes the TATA box
      • TFⅡA
        • Stabilizes binding of TFIIB and TBP to the promoter
      • TFⅡB
        • Binds to TBP; recruits Pol II–TFIIF complex; accurately positions pol II at the TSS;
      • TFⅡD
        • Required for initiation at promoters lacking a TATA box
      • TFⅡE
        • Recruits TFIIH; regulate its ATPase and helicase activities
      • TFⅡF
        • Binds tightly to Pol II; binds to TFIIB and prevents binding of Pol II to nonspecific DNA sequences
      • THⅡH
        • Unwinds DNA at TSS (helicaseactivity);phosphorylates CTD at Ser5 ; releases Pol II from the promoter; recruits nucleotideexcision repair protein
  • The transcription process
    • Initiation
      • 1. Formation of a closed preinitiation complex (PIC)
      • 2.Creating an open complex. TFIIH: DNA helicase
      • 3: RNA strand initiation and promoter clearance.
        • TFIIH: kinase
        • Conformational change in the overall complex initiating transcription.
        • After synthesis of 60 to 70 nt, TFIIE and TFIIH release, and RNA pol II enters the elongation phase of transcription
    • Elongation
      • nuclear membrane
        • transcription and translation proceed asynchronously
      • chromatin
        • nucleosome depolymerization
      • Transcription elongation promoted by the RNA pol II
    • Termination
      • Coupled with posttranscriptional modification
      • The site of cleavage/polyadenylation: an upstream AAUAAA motif and a downstream U-rich or GU-rich element
  • Post-transcriptional processing and degradation of eukaryotic RNA
    • 1. Processing of hn-RNA/pre-mRNA to produce mature-mRNA
      • Capping on the 5ʹ end
        • Function of Cap
          • Bind with cap-binding complex of protein(CBC).
          • Prevent from degradation by the 5′ to 3′ exonuclease.
          • Participates in exportation from the nucleus.
          • Participates in translation initiation.
        • The formation process
      • Polyadenylation of the 3ʹ end
        • Function of polyA
          • Bind with polyA-binding proteins (PABP).
          • Prevent from degradation by the 3′ to 5′ exonuclease.
          • Participates in exportation from the nucleus.
          • Participates in translation initiation.
        • The formation process
      • Eukaryotic pre-mRNA splicing
        • Split gene
          • It is the eukaryotic structural gene on which the coding regions (exons) are often interrupted by the noncoding regions (introns). After splicing of introns and connecting exons again, it become mature RNA.
        • Introns
          • • Introns are the intervening nucleotide sequences in the structural gene and its pre-RNA that are removed from the primary transcript when it is processed into a mature RNA
        • Exons
          • Exons are the sequences that are presented in split genes and mature RNA molecules, and they encompass not only protein-coding genes but also the genes for various RNA (such as tRNAs or rRNAs)
        • Pre-mRNA splicing
          • snRNA
            • U1,U2,U4,U5,U6
            • rich in uridine, 100-300nt
          • snRNPs
            • snRNA+proteins
            • involved in mRNA splicing
          • Splicing mechanism of pre-mRNA in spliceosome
            • The U1 snRNA base pairs with 5’ splicing junction.
            • The U2 snRNA forms base pairs with the A site.
            • The U4/U6-U5 “triple” snRNP positions the
            • The U4/U6-U5 “triple” snRNP positions the appropriate portions of the hnRNA for the following splicing reaction
            • Rearrangement of snRNP to form active spliceosome
            • Splicing of introns
            • Introns are removed as lariat
        • Other processing in precursor mRNA
          • Alternative cleavage & polyadenylation site
          • Alternative splicing
          • Alternative cleavage & polyadenylation site + Alternative splicing
    • 2. post-transcriptional processing of rRNA
      • eukaryotic 45S precursor rRNA into three rRNAs.
        • RNaseP
        • RNaseD/Z
        • 子主题
    • 3. post-transcriptional processing of tRNA
      • Cleavage, addition of CCA-OH at 3’end
      • Base modification
      • Splicing (enzyme-catalyzed)