RNA Synthesis, Processing, &

Modification

Prof. Abdulla Bashein

2019-2020
Department of Biochemistry
Faculty of Medicine
University of Tripoli References
Harper’s 31th
Lippincott Ed
6th Ed
 OBJECTIVES
• Describe the molecules involved and the mechanism of
RNA synthesis.
• Explain how eukaryotic DNA-dependent RNA polymerases,
in collaboration with an array of specific accessory factors,
can differentially transcribe genomic DNA to produce
specific messenger RNA (mRNA) precursor molecules.
• Describe the structure of eukaryotic mRNA precursors,
which are highly modified internally and at both termini.
• Appreciate the fact that the majority of mammalian mRNA
encoding genes are interrupted by multiple nonprotein
coding sequences termed introns, which are interspersed
between protein coding regions termed exons.
 OBJECTIVES
• Explain that since intron RNA does not encode
protein, the intronic RNA must be specifically and
accurately removed in order to generate
functional mRNAs from the mRNA precursor
molecules in a series of precise molecular events
termed RNA splicing.
• Explain the steps and molecules that catalyze
mRNA splicing, a process that converts the end-
modified precursor molecules into mRNAs that
are functional for translation.
Classes of Eukaryotic RNA
RNA IS SYNTHESIZED FROM A DNA TEMPLATE BY
AN RNA POLYMERASE

Genes can be transcribed off both strands of DNA

Some important definitions: Nontemplate “coding” or
“sense” strand
+1
5’- promoter --AGAACTAGATC------3’
3’- --TCTTGATCTAG------5’
RNA: 5’AGAACUAGAUC---
“template” or “anti-sense”
strand
- The “coding” strand of a gene has the same sequence as the RNA
produced
- The RNA polymerase reads the other (template) strand
- RNA polymerases (unlike DNA polymerases) do NOT require a
primer and they cannot proof-read: so errors are likely
- The first nucleotide has a 5’ triphosphate
- Usually RNAs start with a 5’ppp-A or 5’ppp-G

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• A transcription unit is defined as that region
of DNA that includes the signals for
transcription initiation, elongation, and
termination.
• The RNA product, which is synthesized in the
5′–3′ direction, is the primary transcript.
• Transcription frequency varies from gene to
gene but can be quite high.
 Transcription
• Synthesis of RNA in a 5’ to 3’ direction from a
DNA template.
• First step in gene expression
• Needs dsDNA template, RNA polymerase
(RNAP), NTPs & Mg2+
 Transcription
A General Overview
- Mechanisms of transcription in Prokaryotes and Eukaryotes are
basically similar, but a little more complicated / sophisticated in
eukaryotes
-Gene control is carried out primarily by DNA binding proteins that
affect transcription
-Bacteria have no nuclei
 mRNA, once generated is completely accessible to ribosomes
Since both TRANSCRIPTION & TRANSLATION occur 5’  3’,
bacterial protein synthesis can proceed on an mRNA even as it is
being synthesized- in other words, the two processes are coupled

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Concurrent Synthesis of mRNA &
Proteins in Bacteria
 In general there are three elements of gene
control in prokaryotes
• 1. Transcriptional initiation - Most Important
• 2. Transcriptional Elongation
• 3. Transcriptional Termination - significant effects.
• - Rapid RNA Turnover - Important
• - Bacterial mRNAs are not chemically modified before
translation (eukaryotic mRNAs are spliced, 5’-capped
& polyadenylated –the major modifications) - The lack
of modifications allows coupled transcription and
translation to occur- this linkage can provide extra
dimensions to gene regulation
• In eukaryotes, transcription occurs in the
nucleus, where no ribosomes are present –
although ribosomal precursors mature in the
nucleolus –
• mRNAs also requires covalent modification
before emerging into the cytoplasm
• Coupled transcription and translation cannot
occur.
• Single cell - multicellular organism with
differentiated tissues
• Prokaryotes & Eukaryotes also differ in the
apparent use of gene control
• Bacteria: gene-control allows a single cell to
adjust rapidly to changing environmental
conditions (may explain why prokaryotic
mRNAs are short-lived)
• Eukaryotes (especially metazoans): Some
genes serve the above function; But another
very important role for gene control is
regulation of complex genetic programs
 DNA-Dependent RNA Polymerase
Binds to a Distinct Site, the Promoter,
and Initiates Transcription
 Bacterial DNA-Dependent RNA
Polymerase Is a Multisubunit Enzyme
•The core RNA polymerase, ββ’α2ω, often termed E, associates
with a specific protein factor (the sigma [σ] factor) to form
holoenzyme, ββ α2ωσ, or Eσ.
•The σ subunit enables the core enzyme to recognize and bind
the promoter region to form the preinitiation complex (PIC).
• β subunit binds Mg++ ions and composes the
catalytic subunit
Prokaryotic promoters share two regions of
highly conserved nucleotide sequence
 Two lines of evidence define key elements in prokaryotic promoters:
I. Consensus
elements from
sequencing
mRNA data

nucleotide
nomenclatur
-1 +1 +2 +3 e
+1 = first
15-20 5-8 transcribed
18
nucleotide
• The predominant bacterial transcription
termination signal contains an inverted,
hyphenated repeat (the two boxed areas)
followed by a stretch of AT base pairs
Overview of Transcription
The transcription cycle
•- RNA polymerase holoenzyme scans DNA (tracks
along DNA)
•- Binds promoter to give a “closed” complex
•Footprinting reveal that RNA polymerase protects
nucleotides from –55 to +5 from nucleases.
•- ‘Melts’ ~18 bp around “-10” region ( “open”
complex)
•- RNA synthesis begins at “+1” (transcription start
site (TSS))
•- At the start of synthesis, σ is released.
 Elongation
- does not require  factor
- a “Transcription Bubble” migrates down the DNA
~18bp of melted DNA
~8bp of this paired with nascent RNA
~1 turn of an ‘A-type’ helix
- no more than this to prevent RNA from
getting entwined in the DNA
The RNA-DNA mini-helix can rotate about the DNA
strand to constantly free the nascent transcript
- elongation moves at ~50 bases/sec.
- No editing (unlike DNA synthesis)
Accuracy: ~ 1 error in 105 bases
Since mistakes are not passed on to daughter cells,
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they are not critical
 The Transcription Bubble

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 Termination:
Two Types:
Rho-Independent or Factor Independent .1
Rho-Dependent .2

Rho-Independent: Involves formation of a G=C rich hairpin in the 1)

nascent RNA strand, followed by a run of Us

The hairpin is thought to promote RNA dissociation from the template

The poly U sequences leaves a weak residual association
 RNA falls off

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Rho-Independent: Involves formation of a G=C rich hairpin in the nascent RNA
strand, followed by a run of Us
The hairpin is thought to promote RNA dissociation from the template
The poly U sequences leaves a weak residual association
 RNA falls off
 “Hairpin”

Weak association
A:U base pairs are weaker than G:C’s.

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 Rho-Dependent: 1)
“Rho” is a bacterial Termination Factor
It acts as a hexamer of 46 kD subunits
- binds a specific 72 base sequence of ssRNA
It then hydrolyses ATP and eventually disrupts
pairing between the nascent strand & template

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 Bacterial DNA-Dependent RNA
Polymerase Is a Multisubunit Enzyme
The core RNA polymerase, ββ’α2ω, often •
termed E, associates with a specific protein
factor (the sigma [σ] factor) to form
holoenzyme, ββ α2ωσ, or Eσ.
The σ subunit enables the core enzyme to •
recognize and bind the promoter region to
form the preinitiation complex (PIC).
 Mammalian Cells Possess Three Distinct
Nuclear DNA-Dependent RNA Polymerases

α-amanitin ia a peptide toxin from the mushroom Amanita phalloides

They all have two large subunits, that have strong sequence
similarities to prokaryotic β and β′ subunits, and a number of
smaller subunits
 EUKARYOTIC RNA

differences between prokaryotes and 

eukaryotes

1. RNA synthesis

2. RNA processing

RNA processing in eukaryotes only 

1° RNA transcripts processed before transport 

to cytoplasm ...
 EUKARYOTIC RNA

transcription mediated by RNA pol 

early in transcription, guanyltransferase adds 7'-methylguanosine “cap” to 5' end of mRNA 
EUKARYOTIC RNA

AAUAAA sequence near 3' end initiates cleavage ... 

by endonuclease ~ 20 bp downstream 
 EUKARYOTIC RNA

poly(A) polymerase adds poly(A) tail of 150-200 adenosine residues to 3' end cleavage site 
complete °1 mRNA 
 EUKARYOTIC RNA

°1 mRNA shortened before transport to cytoplasm 

chicken ovalbumin DNA/mRNA hybrid  
coding sequences... exons 
intervening sequences... introns... 
introns spliced from mRNA 
genomic DNA  RNA exons + introns 
 EUKARYOTIC RNA

summary... 
genomic DNA  RNA exons + introns 
transcribed  1° mRNA transcript 
processed  cap & polyadenylation 
spliced  splicing intermediate 
spliced  mature mRNA 
 EUKARYOTIC RNA

splicing reaction 
intron lariat structure 
introns excised by 2 transesterification reactions 
 EUKARYOTIC RNA

1 gene  multiple functions by alternative splicing 

multiple gene functions in different... 

tissues 
developmental stages 

different mRNAs from same 1° mRNA transcript... e.g., -tropomyocin gene... 

 EUKARYOTIC RNA

1 gene  multiple functions by alternative splicing e.g., -tropomyocin gene... 

EUKARYOTIC RNA

gene splicing mechanism... 

sequence homologies at exon-intron splice junctions 
= consensus sequences for splicing enzymes... 
Most introns start from the sequence GU and end with •
the sequence AG (in the 5' to 3' direction).
They are referred to as the splice donor and splice •
acceptor site, respectively. However, the sequences at
the two sites are not sufficient to signal the presence
of an intron.
Another important sequence is called the branch •
site located 28-37 bases upstream of the acceptor site.
The consensus sequence of the branch site is
"CU(A/G)A(C/U)", where A is conserved in all genes.
In over 60% of cases, the exon sequence is (A/C)AG at •
the donor site, and G at the acceptor site.
 DNA and RNA synthesis do differ in
several important ways
• Ribonucleotides are used in RNA synthesis rather than
deoxyribonucleotides;
• U replaces T as the complementary base for A in RNA;
• A primer is not involved in RNA synthesis as RNA
polymerases have the ability to initiate synthesis de
novo;
• In a given cell only portions of the genome are
vigorously transcribed or copied into RNA, whereas the
entire genome must be copied, once and only once
during DNA replication; and
• There is no highly active, efficient proofreading
function during transcription.