Part 1

RNA Transcription
Biomedical Sciences for Optometry I
(OPT 2034)

Dr. Christinal Teh

christinaltehpw@segi.edu.my
 Learning Objectives & Outcomes

• At the end of this lecture, all students should be able

to discuss the
1. RNA classification & transcription √
2. Transcriptional control of gene expression
 Central dogma

• Central dogma explains the flow of genetic information from DNA to RNA, RNA
to polypeptides
• 3 main processes: DNA replication, Transcription and Translation
 RNA
• Ribo-nucleic acids
• 3 major types: rRNA (80%), tRNA
(15%) and mRNA (5%)
• Polymer of nucleoside
monophosphate
• Contain ribose instead of
deoxyribose (in DNA)
• The base; Thymine is replaced with
uracil
• RNA are capable to fold to form
secondary structures  therefore
different in size, function and
structure
T is changed to U in RNA
 Structure of RNA

3 types
Ribosomal RNA Transfer RNA
(rRNA) (tRNA)

Messenger RNA
(mRNA)
 rRNA

• rRNA : serve as a sites for protein synthesis.

• Size of these ribosomes varies:-
Prokaryotic cells: 5S, 16S, 23S
Eukaryotic cells: 5S, 5.8S, 18S, 28S

S denotes Svedberg unit – related to the molecular

weight and shape of the subunit itself
• rRNA make up about 80 % of total RNA in the cell.
 tRNA
• Smallest among the 3 (4S only).
• Fx: to carry amino acid to ribosome for
protein synthesis (on the 3’ end)
• Forms secondary structures (D-loop) due to
pairing of complimentary regions within
itself
• Special feature: recognition site at the
anticodon; which will recognize the codons
on the mRNA (complimentary)
• 15 % of tRNA in the cell
 mRNA
• Fx: Carries genetic information from the DNA to the
cytosol; template for protein synthesis
• 5% of mRNA in the cell
• Two types:
Polycistronic (prokaryotes): > 1 gene
Monocistronic (eukaryotes): 1 gene only
• Special structural features
(eukaryotic):-
1. Poly-A tail (3’)
2. Capping (5’)
 Overview
• Transcription for PROKARYOTIC genes
Initiation
Elongation
Termination
• Transcription for EUKARYOTIC genes
Initiation
Elongation
Termination
• Post-transcription MODIFICATIONS
 The RNA polymerase
• This polymerase synthesize all of the RNAs except for
the primers in DNA replication (primase)
• Multi-sub-unit enzyme: , β’, β, σ, Ω
• RNA is syn: 5’3’ direction, antiparallel
• In RNA, T (thymidine) is replaced by U (uridine)
A = U; C Ξ G
• Only 1 of the DNA strand is the template (unlike DNA
synthesis) – determined by the location of the
promoter
 Core enzyme and Holoenzyme
• The RNA polymerase is built up of several multi sub
units
• Core enzyme: 2 Alphas () : enzyme assembly, 1 beta
(β): template binding, 1 beta prime (β’) 5’→3’ RNA
polymerase activity
• Holoenzyme: Core enzyme + subunit sigma (σ)
• (σ) is responsible for promoter recognition
• Fx of the omega subunit (Ω) is unknown
 RNA|Prokaryote| INITIATION
• Binding of RNA Pol to DNA region = promoter
this region has “consensus” sequences, not transcribed
• Recognized by sigma factor
• Mutation: either pribnow/-35 sq  affects transcription
• No need primer (compared to DNA syn)
Down
Upstream Stream

2 1 3
No zero
 RNA|Prokaryote| ELONGATION
• Holoenzyme bound to the promoter region, unwind the DNA helix,
begins transcription
• The unwinding cause supercoils (relieved by DNA topoisomerase);
Sigma factor leaves (after 10 bp); core factor leaves the promoter;
move along the template strand
• Short DNA-RNA helix is formed
• No primer and no proofreading
 RNA|Prokaryote| TERMINATION

• Termination factor : spontaneous (rho

independent) or rho (ρ) dependent
• (1) rho independent
(a) GC- rich region  nascent (new)
RNA forms hairpin
(b) followed by a string of Us at 3’ end
 weak
 detach from DNA template
 DNA template folds back
• (2) rho dependent
rho (protein); a hexameric ATP
(6); binds to C rich region near
3’ end of nascent RNA
- ATPase activity  moves along
nascent RNA and reached RNA
pol Dissociate RNA pol
- ATP-dependent helicase
activity  separates the
RNA/DNA hybrid
 RNA Transcription|Eukaryote

• More complicated compared to prokaryote, eg.

1. Diff polymerase for each rRNA, tRNA, mRNA
2. Transcription factors, elongation factors
 TF bind to the DNA, required for transcription
complex to form

 Each eukaryotic RNA pol requires its own promoters

and transcription factors
 Histones blocking the way
• In eukaryotes, the DNA is bound to histones proteins, for
stabilization (chromatin)
• These histones blocks the accessibility of RNA pol ; need
chromatin remodelling
• They are acetylated (by E: histone acetylase) to open for
transcription. Reversed by histone deacetylase.

Chromatin remodeling
Active ↔ inactive
chromatin

Euchromatin – region with

active transcribed genes

Heterochromatin – inactive
segments
 Types of RNA pol in Eukaryotic cells

• RNA pol I
Syn the precursor of the rRNA in the nucleolus
• RNA pol II
Syn: precursors to mRNA, small nuclear RNAs (snRNA)
• RNA pol III
Syn: precursors to snRNA, tRNA, rRNA
 RNA|Eukaryotic INITIATION
• Recognition site; Hogness TATA box (-25) and CAAT box
(-70)
• Some “constitutive” genes (always being expressed);
does not require TATA box, but GC rich region
• These are sites for Transcription Factors (TF) binding
 New terms: Elements
• Cis-acting elements
Elements that promote transcription that are from the
same molecule of DNA
Eg: GC rich region, promoter sequence

• Trans-acting element
Elements that promote transcription that are from a
different region/gene DNA
Eg. Transcription factors
Role of enhancers in eukaryotic gene regulation
• Enhancers (cis) are DNA sequences that
increases the rate of initiation of
transcription
a) they can be upstream/downstream
b) close or far (1k bases)
c) either strand of DNA (same
chromosome)
• These sequences has “response
elements” which binds to specific TF that
stimulate the transcription process
• Same thing, in reverse: “silencers” 
reduce the level of expression
 α-amanitin

• Inhibitor for RNA polymerase III

• Potent toxin produce by the poisonous mushroom
Amanita phalloides (sometimes called death cap).
• It forms tight complex with polymerase, inhibit mRNA
synthesis and ultimately protein synthesis.
 Post-transcriptional modifications
1. rRNA
2. tRNA
3. mRNA (not much modification in prokaryotic)
a) 5’ capping
b) Poly A tail
c) Removal of introns
 Post-transcription mod: rRNA
• Both prokaryotic and eukaryotic
• Precursor RNAs are cleaved
• E: ribonucleases
 Post-transcription mod: tRNA
• Both prokaryotic and eukaryotic
• Intron remove to form the anticodon
• 3’ and 5’ trimmed
• ‘3 end is added with –CCA (E: nucleotidyl-transferase)
• Base modification to form “unusual” bases (bases
other than A,U,C,G)
 modified tRNA interaction with ribosomes and
anticodon base-pairing properties
tRNA has a CCA head, anticodon & mod-bases
Post-transcription mod: mRNA - Capping
• The cap is 7-methylguanosine attached to 5’ end of mRNA
• The addition is catalyzed by: guanylyl-transferase
• Methylation: by guanine-7-methyl transferase in cytosol
(methyl group from s-adenosyl-methionine)
• Cap added during transcription; stabilizes
• mRNA lack cap, not efficiently translated
Post-transcription mod: mRNA – Poly A tail
• At 3’ end of mRNA, tail is added, not transcribed (E:
polyadenylate polymerase)
• Substrate: ATP
• Fx: stabilize it, facilitate exit to cytosol and translation
• Length: 40-200 adenine nucleotides
Post-transcription mod: mRNA - Splicing

• Spliceosome. Fx: mediate splicing

• Fx: removal of introns which are not translated; in
some tissue  generate variation  produce a
diverse set of proteins from a limited set of genes
• Statistic: Mutation at splice site result in 15% of
all genetic diseases
 Importance of mRNA splicing

• Some pre-mRNA from the same gene can be spliced

differently in different tissues
• This leads to different mRNA product and eventually
different proteins being produced
•  Diverse set of proteins from a limited set of genes
Throw the INTRONS; Express the EXONS