RNA Synthesis

and Splicing
 Transcription: DNA→RNA
DNA (template) + (RNA)n + NTP
 (RNA)n+1 + PPi

STAGES:
1. Initiation

2. Elongation

3. Termination
 Features
Accuracy-RELATIVE
Signals-STOPS AND STOPS
STAGES-Initiation, Elongation and
Termination
E. Coli RNA Polymerase

Initiation
 Holoenzyme: 450kd, 4 subunits
 Sigma subunit: promoter recognition

Elongation
 Core enzyme: RNA chain elongation,
contains catalytic site, is 100% processive
 Transcription is catalyzed by RNA
polymerase

Prokaryotic and Eukaryotic RNA Polymerases:

Conserved structure of largest subunits
Functions of RNA Polymerase
 Search for initiation site: ~2000 promoters

 Unwinds DNA template

 Selects the correct NTP for base pairing initiation

and elongation in RNA synthesis

 Detects termination signals

 Interacts with activator and repressor proteins

(called transcription factors): controls transcriptional
level

 Needs no primers to initiate [ A or G first at 5’ end]

 No error correcting activity: Error rate is 1-0 -4 to -5

core

Sigma: used for initiation only

(2,,’) 
core

holoenzyme
E. coli RNA Polymerase: Roles of
subunits

Subunit Role
 Binds regulatory sequences

 Forms phosphodiester bonds

 Binds DNA template

 Recognizes promoter and initiates

synthesis
RNA polymerase active site. The 3’OH of the growing RNA
chain attacks the -phosphate of the incoming nucleoside
triphosphate.
Transcription is initiated
at promoter sites on the
DNA template
 Promoters:
 DNA sequences that direct RNA
polymerase to the proper
initiation site for transcription

 Identified by DNA Footprinting

 Missing
cuts

Footprinting
 Prokaryotic Start Signals
(Promoter)

Coding strand sequences

Different classes of genes have promoters with variations

of the 2 conserved sequences.
All have different sigma factors for transcription
initiation.
Sigma subunits of RNA polymerase recognize promoter sites

 Subunit has -helix

for -10 sequence
recognition

Most frequent -10 bases

Structure of the  subunit
RNA polymerase must unwind
the template double helix for
transcription to take place
DNA unwinding. RNA polymerase unwinds 17 base
pairs of template DNA.
RNA chains are formed de novo
and grow in the 5’-to3’ direction
 Transcription Reaction
X
X Y

P P P YTP PPi
OH P P P
P
OH

ZTP
X= Purine (G/A)

PPi
5’→3’ Growth X Y Z

P P P
P
P
 Elongation takes place at
transcription bubbles that moves
along the DNA bubbles
 100% Processive

Rate: 50 bases/sec

Transcription bubble: Duplex DNA is unwound at the

forward end of RNA polymerase and rewound at its rear
end. The RNA-DNA hybrid rotates during elongation
RNA-DNA hybrid separation. A structure within RNA
polymerase forces the separation of the RNA-DNA
hybrid, allowing the DNA strand to exit in one direction
and the RNA product to exit in one direction.
 Termination signal

An RNA hairpin 1. Rich in

GC base
followed by
pairs
several uracil RNA transcript
residues
terminates the
transcription of 2. Runs of
some genes U’s
 The rho protein helps terminates the
transcription of some genes

Effect of  protein on the size of RNA transcripts.

Mechanism for the termination of transcription by  protein.
This protein is an ATP-dependent helicases that binds the
nascent RNA chain and pulls it away from RNA polymerase
and the DNA template
Post-Transcriptional Processing of
RNA

Examples

 rRNA precursors

 tRNA precursors

 Modified bases
 E. Coli rRNA transcript: Processed to
create mature rRNAs and tRNA

Many cuts by several ribonucleases

Primary transcript. Cleavage of this transcript

produces 5S, 16S, and 23 S rRNA molecules.
 Enzyme
adds
Modified
bases

Arrows
point to
nuclease
cut sites

Transfer RNA precursor processing. The conversion of a yeast

tRNA precursor into a mature tRNA requires the removal of a 14-
nucleotide intron, the cleavage of a 5’ leader, and the removal of UU
and the attachment of CCA at the 3’ end. In addition, several bases
are modified.
Post-transcriptional base changes
 Bases and ribose are chemically modified
 Many unusual bases are found in tRNA
Unusual bases in tRNA
Inhibitors of transcription

Rifampicin
Actinomycin D
-Amanitin
Rifampicin binds to RNA Polymerase holoenzyme
where DNA-RNA hybrid starts (after 2-3 bases)

 Competes with DNA-RNA hybrid

Inhibits phosphodiester bond formation
Rifamycin: Initiation
(binds to  subunit
and prevents
phosphodiester bond
formation)

Actinomycin D:
Elongation inhibitor
Actinomycin D intercalate between DNA
base pairs
Eukaryotic transcription and
translation are separated in
space and time.
 TRANSCRIPTION AND TRANSLATION:
Prokaryotic and Eukaryotic

COUPLED SEPARATE
COMPARTMENTS
RNA in eukaryotic cells is synthesized by
three types of RNA polymerase
Transcription Promoters:
Prokaryotic vs Eukaryotic
 TBP (30 kD) is
DNA recognition
subunit of TFIID
(700 kD)

Complex formed by TATA-box-binding protein and

DNA. The saddlelike structure of the protein sits atop a
DNA fragment that is both significantly unwound and bent.
 Initiation: Transcription Factor (TF)
Complex Assembly

(30 kD subunit of TFIID)

IIA binds
to IID
and DNA

Assembly of the initiation complex. A ternary complex

between the TATA-box-binding protein, TFIIA, and DNA.
TFIIA interacts primarily with the other protein.
 **
Transcription initiation
complex formation *
*

* Helicase

*Stepwise addition of
TF’s BEFORE start of
transcription
*

*
Initiation of transcription
 More transcription factors that bind to specific sequences

Sp1 protein

HSTF

Transcription factor binding sites. These multiple binding sites for

transcription factors were mapped by footprinting. (A) Binding of Sp1
(green) to the SV40 viral promoter and to the dihydrofolate reductase
(DHFR) promoter (B) Binding of HSTF (blue) to a Drosophila heat-
shock promoter
 Cap

Capping the 5’end. Caps at

the 5’end of the eukaryotic
mRNA include 7-
methylguanylate attached by
a triphosphate linkage to the
ribose at the 5’ end.

one or both
 Transcription Termination and Poly(A)
addition

Polyadenylation of a primary transcript. A specific endonuclease cleaves the

RNA downstream of AAUAAA. Poly(A)polymerase then adds about 250
adenylate residue.
RNA Editing. Enzyme
catalyzed deamination of a
specifc cytidine residue in
the mRNA for
apolipoprotein B-100
changes a codon for
glutamine (CAA) to a stop
codon (UAA).
Apoliporotein B-48, a
truncated version of the
protein lacking the LDL
receptor-binding domain, is
generated by this
postrancriptional change in
the mRNA sequence.
 Eukaryotic mRNA Intron Splicing: 3
Conserved sequence signals

Splice sites. Consensus sequence for the 5’ splice site

and the 3’ splice site are shown. P stands for
pyrimidine.
 (new spliced product results in a
shorter protein sequence)

Splicing defects. Mutation of a single base (G to A) in

an intron of the -globin gene leads to thalassemia. This
mutation generates a new 3’ splice sites akin to the
normal one but farther upstream.
 2

Splicing mechanism used for mRNA

precursors: 2 Transesterifications
 To 3’ of intron

Branch Point:
2’, 3’, 5’-(A) phosphotriester

5’G of intron

Next base

Splicing branch points

 Spliceosome components

(snRNPs) Small Nuclear Ribonucloprotein Particles:

Have RNA and proteins and are involved in splicing
One Role of snRNP RNAs: [example]
U1 RNA base pairs with 5’-splice site
 [SnRNP
Complex]

Spliceosomes: Ordered Assembly

Splicing catalytic center.

U6 and U2 hold
everything else in place

U5 snRNA Branch site

U1 snRNA

U1 and U5 hold exons in place

Alternative splicing: Different mRNAs
from same pre-mRNA