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					 Chapter 11

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              The biochemistry and molecular
              biology department of CMU
          Transcription


The synthesis of RNA molecules using
DNA strands as the templates so that
the genetic information can be
transferred from DNA to RNA.
        Similarity between
   replication and transcription

• Both processes use DNA as the
  template.
• Phosphodiester bonds are formed in
  both cases.
• Both synthesis directions are from 5´
  to 3´.
         Differences between
     replication and transcription

             replication      transcription

template    double strands    single strand

substrate        dNTP             NTP

 primer           yes              no

Enzyme      DNA polymerase   RNA polymerase

product         dsDNA            ssRNA

base pair       A-T, G-C      A-U, T-A, G-C
     Section 1

Template and Enzymes
• The whole genome of DNA needs to
  be replicated, but only small portion
  of genome is transcribed in response
  to the development requirement,
  physiological need and
  environmental changes.
• DNA regions that can be transcribed
  into RNA are called structural genes.
§1.1 Template

 The template strand is the strand
 from which the RNA is actually
 transcribed. It is also termed as
 antisense strand.
 The coding strand is the strand
 whose base sequence specifies the
 amino acid sequence of the encoded
 protein. Therefore, it is also called as
 sense strand.
5'   GCAGTACATGTC       3' coding
                             strand
3'   CGTCATGTACAG       5'   template
                             strand


            transcription


5'   GCAGUACAUGUC       3'   RNA
      Asymmetric transcription
• Only the template strand is used for the
  transcription, but the coding strand is
  not.
• Both strands can be used as the
  templates.
• The transcription direction on different
  strands is opposite.
• This feature is referred to as the
  asymmetric transcription.
5'   3'
3'   5'
Organization of coding information in
the adenovirus genome
§1.2 RNA Polymerase
• The enzyme responsible for the RNA
  synthesis is DNA-dependent RNA
  polymerase.
  – The prokaryotic RNA polymerase is a
    multiple-subunit protein of ~480kD.
  – Eukaryotic systems have three kinds of
    RNA polymerases, each of which is a
    multiple-subunit protein and responsible
    for transcription of different RNAs.
              Holoenzyme

The holoenzyme of RNA-pol in E.coli
consists of 5 different subunits: 2  
.


                                        
  holoenzyme
  core enzyme                      
                                        
                                   
          RNA-pol of E. Coli

subunit    MW             function
                   Determine the DNA to be
         36512
                   transcribed

         150618   Catalyze polymerization

        155613   Bind & open DNA template
                   Recognize the promoter
         70263
                   for synthesis initiation
• Rifampicin, a therapeutic drug for
  tuberculosis treatment, can bind
  specifically to the  subunit of RNA-
  pol, and inhibit the RNA synthesis.
• RNA-pol of other prokaryotic
  systems is similar to that of E. coli in
  structure and functions.
         RNA-pol of eukaryotes

RNA-pol            I           II              III

                                          5S rRNA
products       45S rRNA     hnRNA           tRNA
                                           snRNA

 Sensitivity
                  No          high        moderate
to Amanitin

Amanitin is a specific inhibitor of RNA-pol.
§1.3 Recognition of Origins
• Each transcriptable region is called
  operon.
• One operon includes several structural
  genes and upstream regulatory
  sequences (or regulatory regions).
• The promoter is the DNA sequence that
  RNA-pol can bind. It is the key point
  for the transcription control.
           Promoter


     regulatory
                   structural gene
     sequences
5'                                   3'
        promotor
         RNA-pol
3'                                   5'
           Prokaryotic promoter

5'                                                 3'
     -50    -40   -30   -20    -10    1    10
3'                                                 5'
                   -35
                  region           -10     start
           TTGACA                 region
           AACTGT
                            TATAAT
                            ATATTA
                           (Pribnow box)

           Consensus sequence
          Consensus Sequence




Frequency in 45 samples   38 36 29     40 25 30
                            37 37 28     41 29 44
• The -35 region of TTGACA sequence
  is the recognition site and the
  binding site of RNA-pol.
• The -10 region of TATAAT is the
  region at which a stable complex of
  DNA and RNA-pol is formed.
     Section 2

Transcription Process
          General concepts

• Three phases: initiation, elongation,
  and termination.
• The prokaryotic RNA-pol can bind to
  the DNA template directly in the
  transcription process.
• The eukaryotic RNA-pol requires co-
  factors to bind to the DNA template
  together in the transcription process.
§2.1 Transcription of Prokaryotes

• Initiation phase: RNA-pol recognizes
  the promoter and starts the
  transcription.
• Elongation phase: the RNA strand is
  continuously growing.
• Termination phase: the RNA-pol stops
  synthesis and the nascent RNA is
  separated from the DNA template.
a. Initiation

• RNA-pol recognizes the TTGACA
  region, and slides to the TATAAT
  region, then opens the DNA duplex.
• The unwound region is about 171 bp.
• The first nucleotide on RNA transcript
  is always purine triphosphate. GTP is
  more often than ATP.
• The pppGpN-OH structure remains on
  the RNA transcript until the RNA
  synthesis is completed.
• The three molecules form a
  transcription initiation complex.

  RNA-pol (2) - DNA - pppGpN- OH 3
• No primer is needed for RNA
  synthesis.
• The  subunit falls off from the RNA-
  pol once the first 3,5 phosphodiester
  bond is formed.
• The core enzyme moves along the
  DNA template to enter the elongation
  phase.
b. Elongation

• The release of the  subunit causes
  the conformational change of the
  core enzyme. The core enzyme slides
  on the DNA template toward the 3
  end.
• Free NTPs are added sequentially to
  the 3 -OH of the nascent RNA strand.
  (NMP)n + NTP             (NMP)n+1 + PPi
                            elongated
  RNA strand   substrate   RNA strand
• RNA-pol, DNA segment of ~40nt and
  the nascent RNA form a complex
  called the transcription bubble.
• The 3 segment of the nascent RNA
  hybridizes with the DNA template, and
  its 5 end extends out the
  transcription bubble as the synthesis
  is processing.
Transcription bubble
RNA-pol of E. Coli
RNA-pol of E. Coli
   Simultaneous
transcriptions and
    translation
c. Termination


• The RNA-pol stops moving on the
  DNA template. The RNA transcript
  falls off from the transcription
  complex.
• The termination occurs in either  -
  dependent or  -independent manner.
 The termination function of  factor




The  factor, a hexamer, is a ATPase
and a helicase.
    -independent termination

• The termination signal is a stretch of
  30-40 nucleotides on the RNA
  transcript, consisting of many GC
  followed by a series of U.
• The sequence specificity of this
  nascent RNA transcript will form
  particular stem-loop structures to
  terminate the transcription.
                      rplL protein
DNA
5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3
5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3




RNA

                                               UUUU...…


                     UUUU...…
       Stem-loop disruption
• The stem-loop structure alters the
  conformation of RNA-pol, leading to
  the pause of the RNA-pol moving.
• Then the competition of the RNA-
  RNA hybrid and the DNA-DNA hybrid
  reduces the DNA-RNA hybrid
  stability, and causes the
  transcription complex dissociated.
• Among all the base pairings, the
  most unstable one is rU:dA.
§2.2 Transcription of Eukaryotes
 a. Initiation
 • Transcription initiation needs
   promoter and upstream regulatory
   regions.
 • The cis-acting elements are the
   specific sequences on the DNA
   template that regulate the
   transcription of one or more genes.
           Cis-acting element

      cis-acting element
                                      structural gene
       GCGC      CAAT   TATA
                               exon    intron exon


                                   start
                        TATA box      (Hogness box)

enhancer         CAAT box

        GC box
TATA box
        Transcription factors
• RNA-pol does not bind the promoter
  directly.
• RNA-pol II associates with six
  transcription factors, TFII A - TFII H.
• The trans-acting factors are the
  proteins that recognize and bind
  directly or indirectly cis-acting
  elements and regulate its activity.
TF for eukaryotic transcription
    Pre-initiation complex (PIC)

• TBP of TFII D binds TATA
• TFII A and TFII B bind TFII D
• TFII F-RNA-pol complex binds TFII B
• TFII F and TFII E open the dsDNA
  (helicase and ATPase)
• TFII H: completion of PIC
Pre-initiation complex (PIC)


          RNA pol II

                  TF II F     TF II E
TF II   TBP TAF
                   TF II
 A      TATA        B
                            TF II H     DNA
    Phosphorylation of RNA-pol

• TF II H is of protein kinase activity to
  phosphorylate CTD of RNA-pol. (CTD
  is the C-terminal domain of RNA-pol)
• Only the p-RNA-pol can move toward
  the downstream, starting the
  elongation phase.
• Most of the TFs fall off from PIC
  during the elongation phase.
b. Elongation

• The elongation is similar to that of
  prokaryotes.
• The transcription and translation do
  not take place simultaneously since
  they are separated by nuclear
  membrane.
                          nucleosome

            RNA-Pol

 moving
direction



                      RNA-Pol




                            RNA-Pol
c. Termination


• The termination sequence is AATAAA
  followed by GT repeats.
• The termination is closely related to
  the post-transcriptional modification.
    Section 3

Post-Transcriptional
   Modification
• The nascent RNA, also known as
  primary transcript, needs to be
  modified to become functional tRNAs,
  rRNAs, and mRNAs.
• The modification is critical to
  eukaryotic systems.
§3.1 Modification of hnRNA
• Primary transcripts of mRNA are called as
  heteronuclear RNA (hnRNA).
• hnRNA are larger than matured mRNA by
  many folds.
• Modification includes
   –   Capping at the 5- end
   –   Tailing at the 3- end
   –   mRNA splicing
   –   RNA edition
      a. Capping at the 5- end
                 OH       OH
                                                                     O
                                                                 N
                                                                         NH
                                O        O   O
                      O                              5'
H2N    N     N            H2C O P   O P O P O CH2                N         NH 2
                                                                     N
                           5'                               O
                                O        O   O
  HN
             N
       O
             CH 3
                                                      O         OH
                                    Pi
                                                 O        P O            AAAAA-OH   3'
                                                      O



           m7GpppGp----
                  ppp5'NpNp
                                     removing
                              Pi     phosphate group
                   pp5'NpNp
                               GTP forming 5'-5'
                                       triphosphate group
                               PPi
          G5'ppp5'NpNp

                              methylating at G7

              7
          m       GpppNpNp
                              methylating at C2' of the
                              first and second
                              nucleotides after G
    7
m       Gpppm2'Npm2'Np
• The 5- cap structure is found on
  hnRNA too.  The capping process
  occurs in nuclei.
• The cap structure of mRNA will be
  recognized by the cap-binding protein
  required for translation.
• The capping occurs prior to the
  splicing.
b. Poly-A tailing at 3 - end
• There is no poly(dT) sequence on the
  DNA template.  The tailing process
  dose not depend on the template.
• The tailing process occurs prior to the
  splicing.
• The tailing process takes place in the
  nuclei.
c. mRNA splicing

                         mRNA




           DNA



The matured mRNAs are much shorter than
the DNA templates.
                    Split gene

The structural genes are composed of
coding and non-coding regions that
are alternatively separated.

                             7 700 bp
   L       1    2    3   4              5       6       7
       A       B C D            E           F       G


A~G no-coding region 1~7 coding region
          Exon and intron

Exons are the coding sequences that
appear on split genes and primary
transcripts, and will be expressed to
matured mRNA.

Introns are the non-coding sequences
that are transcripted into primary
mRNAs, and will be cleaved out in the
later splicing process.
mRNA splicing
Splicing mechanism
lariat
Twice transesterification
                       intron


            5'exon                           3'exon
       5'               U pA         G pU                 3'

                                   first transesterification
               pG-OH
                     pGpA

   5'                  UOH           G pU                 3'


                                second transesterification
                                     5' pGpA

  5'                 U pU               3'


                                                 GOH 3'
d. mRNA editing

• Taking place at the transcription
  level
• One gene responsible for more than
  one proteins
• Significance: gene sequences, after
  post-transcriptional modification,
  can be multiple purpose
  differentiation.
 Different pathway of apo B

           Human apo B
              gene


       hnRNA (14 500 base)
                             CAA to UAA
                             At 6666
  liver
apo B100
                          intestine
(500 kD)
                          apo B48
                         (240 kD)
§3.2 Modification of tRNA
       Precursor transcription
DNA


      TGGCNNAGTGC         GGTTCGANNCC


        RNA-pol III




                 tRNA precursor
Cleavage




    RNAase P
  endonuclease

    ligase
Addition of CCA-OH



   tRNA nucleotidyl
     transferase

     ATP   ADP
            Base modification


                      1. Methylation
(2)             (1)      A→mA, G→mG
      (1)
                      2. Reduction
                         U→DHU
                      3. Transversion
                         U→ψ
                (3)   4. Deamination
              (4)        A→I
§3.3 Modification of rRNA

• 45S transcript in nucleus is the
  precursor of 3 kinds of rRNAs.
• The matured rRNA will be assembled
  with ribosomal proteins to form
  ribosomes that are exported to
  cytosolic space.
                                       rRNA


  18S      5.8S          28S            45S-rRNA

                  transcription


                  splicing
18S-rRNA
                             5.8S and 28S-rRNA
§3.4 Ribozyme
• The rRNA precursor of tetrahymena
  has the activity of self-splicing (1982).
• The catalytic RNA is called ribozyme.
• Self-splicing happened often for
  intron I and intron II.
• Both the catalytic domain and the
  substrate locate on the same
  molecule, and form a hammer-head
  structure.
• At least 13 nucleotides are conserved.
Hammer-head
      Significance of ribozyme

• Be a supplement to the central
  dogma
• Redefine the enzymology
• Provide a new insights for the origin
  of life
• Be useful in designing the artificial
  ribozymes as the therapeutical
  agents
     Artificial
    ribozyme
• Thick lines:
  artificial ribozyme
• Thin lines:
  natural ribozyme
• X: consensus
  sequence
• Arrow: cleavage
  point

				
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