Typical Plasmid
Blue/White Selection
Alpha complementation Trick
omega
alpha
Blue/White Selection
Standard 4 exon gene
-Where is the polyA tail in the gene above?
-Draw a 4 exon gene where exon 4 is all 3’UTR
Chapter 11
Transcription and RNA Processing
The Central Dogma
The Central Dogma
The Central Dogma
Transcription and Translation
in Prokaryotes
The primary
transcript is
equivalent to the
mRNA molecule.
The mRNA codons
on the mRNA are
translated into an
amino acid
sequence by the
ribosomes.
Transcription and Translation
in Eukaryotes
 The primary transcript
(pre-mRNA) is a
precursor to the mRNA.
 The pre-mRNA is
modified at both ends,
and introns are
removed to produce the
mRNA.
 After processing, the
mRNA is exported to
the cytoplasm for
translation by
ribosomes.
Types of RNA Molecules
 Messenger RNAs (mRNAs)—intermediates that
carry genetic information from DNA to the ribosomes.
 Transfer RNAs (tRNAs)—adaptors between amino
acids and the codons in mRNA.
 Ribosomal RNAs (rRNAs)—structural and catalytic
components of ribosomes.
 Small nuclear RNAs (snRNAs)—structural
components of spliceosomes.
 Micro RNAs (miRNAs)—short single-stranded RNAs
that block expression of complementary mRNAs.
RNA Synthesis And Transport
in Eukaryotes
Method: PulseChase Labeling
At first, labeled RNA
is exclusively in the
nucleus.
Later, the labeled
RNA is found in the
cytoplasm.
General Features of RNA Synthesis
 Similar to DNA Synthesis except
– The precursors are ribonucleoside triphosphates
ATP, GTP, CTP, UTP.
– Only one strand of DNA is used as a template.
– RNA chains can be initiated de novo (no primer required).
The RNA molecule will be complementary to
the DNA template (antisense) strand and
identical (except that uridine replaces
thymidine) to the DNA nontemplate (sense)
strand.
 RNA synthesis is catalyzed by RNA polymerases and
proceeds in the 5’3’ direction.
Awful representation
Correct Representation of DNA
RNA make a new “top strand”
Nucleophilic attack
A Triplex of Sorts
Prokaryotes-transcription and
translation occur at the same
time
Transcription—the first step in gene
expression—transfers the genetic
information stored in DNA—genes—into
messenger RNA molecules that carry the
information to the ribosomes—the sites of
protein synthesis—in the cytoplasm.
Transcription is terminated in
Prokaryotes
E. Coli RNA Polymerase
Tetrameric core: 2 ’
Holoenzyme: 2 ’ 
Functions of the subunits:
 : assembly of the tetrameric core
 : ribonucleoside triphosphate binding site
 ’: DNA template binding region
 : initiation of transcription
Initiation of RNA Chains
1. Binding of RNA polymerase holoenzyme to
a promoter region in DNA
2. Localized unwinding of the two strands of
DNA by RNA polymerase to provide a
single-stranded template
3. Formation of phosphodiester bonds
between the first few ribonucleotides in the
nascent RNA chain
Numbering of a Transcription Unit
The transcript initiation site
is +1.
Bases preceding the initiation site are given
minus (–) prefixes and are referred to as
upstream sequences.
Bases following the initiation site are given
plus (+) prefixes and are referred to as
downstream sequences.
A Typical E. coli Promoter
Consensus sequences: -10 sequence and 35 sequence
Recognition sequence: -35 sequence
Termination Signals in E. coli
Rho-dependent terminators—require a
protein factor ()
Rho-independent terminators—do not
require 
Rho-Independent Termination
Transcription and RNA
Processing in Eukaryotes
Three different enzymes catalyze transcription
in eukaryotes, and the resulting RNA transcripts
undergo three important modifications, including
the excision of noncoding sequences called
introns. The nucleotide sequenced of some
RNA transcripts are modified
posttranscriptionally by RNA editing.
Modifications to Eukaryotic
pre-mRNAs
A 7-Methyl guanosine cap is added to the 5’
end of the primary transcript by a 5’-5’
phosphate linkage.
A poly(A) tail (a 20-200 nucleotide
polyadenosine tract) is added to the 3’ end of
the transcript. The 3’ end is generated by
cleavage rather than by termination.
When present, intron sequences are spliced
out of the transcript.
Eukaryotes Have Three RNA
Polymerases
Pol II is the only Polymerase that is routinely studied.
Pol I and Pol III are very complicated.
A Typical RNA Polymerase II
Promoter
Initiation by RNA Polymerase II
The 7-Methyl Guanosine
(7-MG) Cap
The 3’ Poly(A) Tail
AATAAA
Interrupted Genes in Eukaryotes:
Exons and Introns
Most eukaryotic genes contain noncoding
sequences called introns that interrupt the
coding sequences, or exons. The introns are
excised from the RNA transcripts prior to their
transport to the cytoplasm.
Removal of Intron Sequences
by RNA Splicing
The noncoding introns are excised
from gene transcripts by several
different mechanisms.
Excision of Intron Sequences
Splicing
Removal of introns must be very precise.
Conserved sequences for removal of the
introns of nuclear mRNA genes are minimal.
– Dinucleotide sequences at the 5’ and 3’ ends of
introns.
– An A residue about 30 nucleotides upstream from
the 3’ splice site is needed for lariat formation.
Types of Intron Excision
The introns of tRNA precursors are excised by
precise endonucleolytic cleavage and ligation
reactions catalyzed by special splicing
endonuclease and ligase activities.
The introns of nuclear pre-mRNA (hnRNA)
transcripts are spliced out in two-step reactions
carried out by spliceosomes.
The Spliceosome
 Five snRNAs: U1, U2,
U4, U5, and U6
 Some snRNAs associate
with proteins to form
snRNAs (small nuclear
ribonucleoproteins)