Aris Kaksis Riga University docent - Chemistry

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)

DNA	RNA
2'-de-oxy-ribose	D-ribose

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).

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
5'———

GACA———————

————••••••••••••••••••••••••••••••••••••3'
                            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

      G
                             |          |

      G
                               \      /
                               G•C
                               A•

                               C•G
                               C•G
                               G•C
                               C•G
                               C•G
                               G•C
   5'-

•C•C•C•A•G| |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———

A box———••••••••••••••••••••••••••••••3'
Promoter sequence Start site Coding sequence

•

A 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

A 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
Initiation
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

A +1 Inr
3'

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