Biochemistry of Medicinals I Phar 6151 CHAPTER THREE
Instructor: Dr. Natalia Tretyakova, Ph.D.
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor

RNA Synthesis
I.          Introduction
While DNA is used for information storage, RNA is the means by which this information is articulated in the organism.  It is in essence the vehicle that translates the DNA code into strings of amino acids AA
                                                                                               (i.e. proteins). There are three 3 types of RNA;
mRNA = Message RNA; a RNA copy of the DNA sequence (gene) used a template for protein synthesis
tRNA = Transfer RNA; a small RNA 76 bp
                                   that is attached to an amino acid AA which can be added to a growing peptide chain
rRNA = Ribosomal RNA; component of ribosomes with catalytic and structural function;
                                                            involved in protein synthesis; three 3 types exist 23S, 16S, and 5S
DNA and RNA differ in several important ways:
1.) RNA is composed of   and not  
2.) The sugar composition of RNA is composed of ribose and not de-oxy-ribose
3.) Sugar pucker on RNA is 3’ endo and RNA is usually single-stranded
     (although it forms hairpins by folding over the same strand)
Quantitiy of RNA in the bacteria E. coli
Type Relative amount, % Sed. Coeff. Sv Mass, kD # of Nucleotides
rRNA 80 23 1.2•103 3700
    16 0.6•103 1700
    5 3.6•101 120
tRNA 15 4 2.5•101 75
mRNA 5   heterogeneous  
II.      RNA Synthesis in Prokaryotes
B.      RNA Polymerases
The synthesis of RNA is catalyzed by RNA polymerases.  They function by catalyzing the
step-by-step addition of ribonucleotides to the 3' end of a RNA polymer chain. 
These enzymes are important for the production of mRNA, tRNA and rRNA from a DNA template.
1.  Polymerization Reaction
Like all polymerization reactions, the RNA synthesis is characterized by three 3 stages;
                                                                                        initiation, elongation, and termination.
RNA polymerization requires:
     Double stranded DNA template strand; rarely does ssDNA serve as template
     Does not need a primer: just GTP or ATP
     Ribonucleoside 5'-triphosphates, ATP, GTP, TP and CTP (NTPs)
     Mg2+ to activate the NTPs
          RNAn bases + NTP  RNAn+1 + HO-O2P-O-PO-2OH
RNA polymerase does the following;
   Searches DNA and finds initiation (promotor) sites
   Unwinds a short stretch of double-helical DNA to produce a ssDNA template
   Adds one 1 NTP after another by the nucleophilic attack of the 3'- hydroxyl -OH on the alpha
a phosphate of the NTP; is totally processive; one enzyme makes the entire transcript (mRNA).
   Addition of NTPs follows Watson-Crick base pairing between the DNA template and the
incoming NTPs;
   Unlike DNA Polymerases, RNA Polymerases do not have nuclease activity;
not able to proof read for mismatches.
   Detects termination signals
   Activator and repressor proteins modulate the rate  of transcription by RNA Pol.
The sequence of the newly synthesized RNA is complementary to the sequence of its
template DNA (antisense strand) and is identical to the sequence of the coding (sense) strand
                                                                                            (with the exception of s instead of s).
5' … A  G G C C  G G A C   C A… 3' Sense strand of DNA
3' …  A C C G G A C C  G A A G … 5' Anti-sense strand of DNA
                                        ¯ Transcription of anti-sense strand
5' … A  G G C C  G G A C   C A… 3' mRNA
                                        ¯ Translation of mRNA 
            Met-Ala-Trp-Thr-Ser- Peptide
2.       RNA Polymerase Structure
RNA polymerases from bacteria are very large (450kD) and are composed of four 4 kind of subunits:
a2bb  (holoenzyme). 37+37+151+155+70=450
Subunit Number Mass kD Function
a 2 37 Binds regulatory sequences
b 1 151 Bonds NTPs, forms phosphodiester bonds
b' 1 155 Binds DNA template
holoenzyme 1 70 Recognizes promoter and initiates synthesis
B.      RNA Replication
Transciption or RNA synthesis occurs along one of the strands of DNA and proceeds 5’®3’.
The DNA strands are locally unwound and separated (replication bubble, about 1.6 turns of B-helix).
1.       Initiation
DNA sequences called promoters determine where transcription begins.  RNA polymerase will bind to these sites in the absence of ribo-nucleoside tri-phosphates.  Common motifs have been observed for these sequences:
Consensus Prokaryotic Promoter Sequence
                    -35                                        -10                   +1
 ­                            Promoter sequence            Start site ­    Coding sequence              ­
           The efficiency of the promoter is measured as the amount of transcription per unit time.  The more transcripts made the better the promoter.  The best promoters are ones that are as close to the consensus sequence as possible.  Strong promoters result in frequent transcription initiation (e.g. 2/sec). Mutations in the promoter region can severely limit a promoters function.
  a2bb  (holoenzyme) is necessary for initiation.  The 5 subunit is necessary for increasing the specificity for
                                                                                 the promoter 10 000 fold over a random DNA sequence.
- Promoter sequence found by sliding of the holoenzyme along DNA duplex until it finds the right
                                   H-bond motif. This takes place without unwinding the double helix.
- Rate of  = 1010 M-1s-1 for binding to promoter sequence.
- Different  subunits for different promoters (i.e., heat shock)
  Before RNA synthesis can begin, RNA Polymerase unwinds a 17 bp reading area of duplex DNA by 1.6 turns to form open promoter complex. This imparts positive (+) DNA supercoiling that can be relieved by
topo-isomerases. Negative (-) supercoiling of DNA can  promoter efficiency by facilitating DNA unwinding.
RNA polymerase needs no primer, however, a molecule of GTP or ATP is used to start transcription.
                                              Mechanism of Nucleotide Addition

2.       Elongation
Elongation after the addition of the first base to GTP or ATP proceeds quickly until termination signal is encountered. 
     The holoenzyme, a2bb   loses the   subunit
     A transcription bubble is formed
     Contains a RNA-DNA duplex of 12 bp
     the RNA Pol locally melts the DNA duplex followed by rewinding
                                                                 (the transcription bubble does not increase ­ in size)
     Rate of  = 50 NTP's per sec.
     Mistakes 1 in 10 000 -100 000 (no proofreading since no nuclease activity)
RNA Polymerization Reaction

                                                                                                ¯ RNA Polymerase 

                                                              17 bp ¯ unwound
                                                                 s ¬¯ NTP's                      ¯¯ Coding strand

                           5'ppp ­                              RNA-DNA ­ 12 kb ­ Template strand
3.           Termination
During the last phase of synthesis, the polymerization reaction is stopped by a specific signal, the RNA-DNA complex comes apart, the melted DNA rewinds, and the RNA polymerase dissociates from the DNA. There are two 2 mechanism for termination.
1.)   Formation of a stable hairpin from palindromic GC rich-region followed by A-rich region. The enzyme pauses after this and the RNA dissociates from the template and enzyme complex.
                                /-C-\                                 RNA Hairpin
                             |          |
                               \      /
   5'-CCCAG|     |A-3'
2.)        The Rho (r) protein factor is a hexamer of identical 419-residue subunits. It recognizes certain regions of RNA that come before the termination site and wraps around them in an ATP driven ® process. This them allows the RNA to be pried off of the RNA-DNA duplex.


III.       RNA Synthesis in Eukaryotes
Unlike prokaryotes, eukaryotes transcription and translation takes place in different compartments. Consequently, control of this processes is much more ­ elaborate.  There are three 3 big differences between prokaryotic and eukaryotic transcription.
            1.)        RNA requires a 5' CAP
            2.)        RNA requires a 3' poly (A) tail.
            3.)        RNA is extensively spliced to remove introns ® off ? RNA.
A.        RNA Polymerases
The synthesis of RNA is catalyzed by three 3 RNA polymerases that all reside in the nucleus.  They function by catalyzing the step-by-step addition of ribo-nucleotides to the 3' end of a
RNA polymer chain.  These enzymes are important for the production of mRNA, tRNA and rRNA from a DNA template.
Type Localization ? RNA produced   Inhibition by amanitin
I Nucleolus rRNA Insensitive
II Nucleoplasm mRNA Strongly inhibits
III Nucleoplasm tRNA,rRNA Weakly inhibits
Amanitin = Octapeptide derived from the Death Cap mushroom
           RNA Pol II is responsible for transcription to mRNA
                 -      8-12 subunits
                 -      Two 2 subunits ( 220 kD and 140 kD) responsible for synthesis
                 -      Regulated by phosphorylation of C-carboxyl-terminal domain (CTD)
                 -      Subunits 5, 6, and 8 are in Type I and II.
      • Same chemical rules that apply for the polymerization reaction for prokaryote synthesis apply to eukaryote                                                                                                                                                        synthesis


B.        RNA Replication
There are many similarities to prokaryotic transcription, but there are also many important differences.
1.         Initiation
Eukaryote promoters are also located upstream ­ from the coding sequence for the transcript.
Consensus Eukaryotic Promoter Sequence
               -110                                  -40                    -25                          +1
5'———CAA box——————GC box———AA box———•••••••••••••••••••••••••••••3'
 ­                            Promoter sequence                                Start site ­        Coding sequence              ­
                       AA box is necessary but not sufficient
                       CAA box and GC box are present but not always
                       Synthesis begins with GTP or ATP
                       5' end is quickly modified to a CAP form with GTP
                              2.      Transcription Factors
RNA polymerase II is unable to initiate synthesis by itself, it must be guided to the start site by a transcription factor (TFII).  This is large family of proteins that interact with RNA Pol II in order to facilitate polymerization.
They bind to the start site  in the following order
            1.)        TFIID; contains AA box binding protein
            2.)        TFIIA
            3.)        TFIIB; binds over start site for coding region
            4.)        RNA Pol II
            5.)        TFIIE
Table 26-1. Proteins Required for Transcription at the RNA Polymerase II Promoters of       Eukaryotes
  Number of    
Transcription factor subunits Subunit MW Functions
RNA polymerase II 12 10 000-220 000 Catalyzes RNA synthesis
TBP (TATA-binding protein)    1 38 000 Specifically recognizes the AA box
TFIIA 3 12 000,19 000,
35 000
Stabilizes binding of TFIIB and TBP to the promoter
TFIIB 1 35 000 Binds to TBP; recruits RNA polymerase-TFIIF complex
TFIID 12 15 000-250 000 Interacts with positive and negative regulatory proteins
TFIIE 2 34 000, 57 000 Recruits TFIIH; ATPase and helicase activities
TFIIF 2 30 000, 74 000 Binds tightly to RNA polymerase II; binds to TFIIB and                          prevents binding of RNA polymerase to
                         nonspecific DNA sequences
TFIIH 12 35 000-89 000 Unwinds DNA at promoter; phosphorylates RNA      polymerase; recruits nucleotide-excision repair complex
Elongation *      
ELL 1 80 000  
P-TEFb 2 43 000,124 000  
SII (TFIIS) 1 38 000  
Elongin (SIII) 3 15 000, 18 000,
110 000
* All Elongation factors suppress the pausing or arrest of transcription by the
                                                                                                   RNA polymerase II-TFIIF complex.
 The name is derived from the term eleven-nineteen lysine-rich leukemia. The gene for the factor ELL is
                                                         the site of chromosomal recombination events frequently associated with
                                                         the cancerous condition known as acute myeloid leukemia.
5' 3'           RNA Polymerase II Complex
            -30 AA      +1 Inr
3' 5'
Other factors may also be necessary depending on the site such as special
                                                                                  transcription factors and enhancers.
                       Enhancers are able to stimulate transcription thousands 1000÷3000 of bp away.
                       Enhancers can be upstream ­, downsteam ¯ and even in the sequence
                       They are generally cell specific.
                       Ex. Enhancer that binds glucocorticoid steroids.
                       Virus can also have enhancers that are tissue and species specific
2.  Termination
Termination of transcription is not well understood.  However, all transcripts end up with a poly (A) tail, which appears to be important for efficient translation.
                       An endonuclease recognizes and cleaves the AA / AA sequence
                       A poly (A) polymerase adds 250 A's to the end.
                       The transcript is transported out of the nucleus