Chapter Eleven

Transcription of the
Genetic Code: The
Biosynthesis of RNA
Transcription
Overview of Transcription
synthesized on a DNA template, catalyzed by DNA-
dependent RNA polymerase
ATP, GTP, CTP, and UTP are required, as is Mg2+
no RNA primer is required
the RNA chain is synthesized in the 5 -> 3 direction;
the nucleotide at the 5 end of the chain retains its
triphosphate (ppp) group
the DNA base sequence contains signals for initiation
and termination of RNA synthesis; the enzyme binds
to and moves along the DNA template in the 3 -> 5
direction
the DNA template is unchanged
Transcription in Prokaryotes
E. coli RNA Polymerase:
molecular weight about 500,000
four different types of subunits: , , , and
the core enzyme is 2
the holoenzyme is 2
the role of the subunit is recognition of the promoter locus;
locus
the subunit is released after transcription begins
of the two DNA strands, the one that serves as the template for
RNA synthesis is called the template strand or antisense
strand;
strand the other is called the coding (or nontemplate) strand
or sense strand
the holoenzyme binds to and transcribes only the template
strand
The Basics of Transcription
Promoter Sequence
Simplest of organisms contain a lot of DNA that is
not transcribed

RNA polymerase needs to know which strand is

template strand, which part to transcribe, and where
first nucleotide of gene to be transcribed is

Promoters-DNA sequence that provide direction for

RNA polymerase
Promoter Sequence
Chain Initiation
First phase of transcription is initiation

Initiation begins when RNA polymerase binds to

promoter and forms closed complex

After this, DNA unwinds at promoter to form open

complex, which is required for chain initiation
Initiation and Elongation in Transcription
Chain Elongation
After strands separated, transcription bubble of ~17
bp moves down the DNA sequence to be transcribed

RNA polymerase catalyzes formation of

phosphodiester bonds between the incorp.
ribonucleotides

Topoisomerases relax supercoils in front of and

behind transcription bubble
Chain Elongation (Contd)
Chain Termination
Two types of termination mechanisms:
intrinsic termination- controlled by specific
sequences, termination sites
Termination sites characterized by two inverted
repeats
Chain Termination (Contd)
Other type of termination involves rho () protein
Rho-dependent termination sequences cause hairpin
loop to form
Transcription Regulation in Prokaryotes
In prokaryotes, transcription regulated by:
alternative factors
enhancers
operons
transcription attenuation

Alternative factors
Viruses and bacteria exert control over which genes
are expressed by producing different -subunits that
direct the RNA polymerase to different genes.
Control by Different Subunits
Enhancers
Certain genes include sequences upstream of
extended promoter region
These genes for ribosomal production have 3
upstream sites, Fis sites
Class of DNA sequences that do this are called
enhancers
Bound by proteins called transcription factors
Elements of a Bacterial Promoter
Operon
Operon: a group of operator, promoter, and
structural genes that codes for proteins
the control sites, promoter, and operator genes are
physically adjacent to the structural gene in the DNA
the regulatory gene can be quite far from the operon
operons are usually not transcribed all the time
-Galactosidase, an inducible protein
coded for by a structural gene, lacZ
structural gene lacY codes for lactose permease
structural gene lacA codes for transacetylase
expression of these three structural genes is
controlled by the regulatory gene lacI that codes for a
repressor
How Does Repression Work
Repressor protein
made by lacI gene
forms tetramer when it
is translated
Repressor protein then
binds to operator
portion of operon
Operator and promoter
together are the
control sites
Binding Sites On the lac operon
Lac operon is induced when E. coli has lactose as
the carbon source

Lac protein synthesis repressed by glucose

(catabolite repression)

E. coli recognizes presence of glucose by promoter

as it has 2 regions: RNA polymerase binding site,
catabolite activator protein (CAP) binding site
Binding Sites On lac operon (Contd)
Catabolite Repression

CAP forms complex with cAMP

Complex binds at CAP site
RNA polymerase binds at available binding site, and
transcription occurs
Basic Control Mechanisms in Gene Control
Control may be inducible or repressive, and these
may be negatively or positively controlled
Control of the trp operon
Trp operon codes for a leader sequence (trpL) and five
polypeptides
The five proteins make up 4 different enzymes that catalyze
the multistep process that converts chorisimate to tryptophan
Alternative 2 structures Can Form in trp
Operon
These structures can
form in the leader
sequence
Pause structure-
binding between
regions 1 and 2
Terminator loop-
binding between
regions 3 and 4
Antiterminator
structure- Alternative
binding between
regions 2 and 3
Attenuation in the trp operon
Pause structure
forms when
ribosome passes
over Trp codons
when Trp levels are
high
Ribosome stalls at
the Trp codon when
trp levels are low
and antiterminator
loop forms
Transcription in Eukaryotes
Three RNA polymerases are known; each
transcribes a different set of genes and recognizes
a different set of promoters:
RNA Polymerase I- found in the nucleolus and
synthesizes precursors of most rRNAs
RNA Polymerase II- found in the nucleoplasm
and synthesizes mRNA precursors
RNA Polymerase III- found in the nucleoplasm
and synthesizes tRNAs, other RNA molecules
involved in mRNA processing and protein transport
RNA Polymerase II
Most studied on the
polymerases
Consists of 12
subunits
RPB- RNA
Polymerase B
How does Pol II Recognize the Correct
DNA?
Four elements of the Pol II promoter allow for this
phenomenon
Initiation of Transcription
Any protein regulator of transcription that is not itself
a subunit of Pol II is a transcription factor

Initiation begins by forming the preinitiation

complex

Transcription control is based here

General Transcription Initiation Factors
Transcription Order of Events
Less is known about
eukaryotes than
prokaryotes
The phosphorylated
Pol II synthesizes
RNA and leaves the
promoter region
behind
GTFs are left at the
promoter or
dissociate from Pol II
Elongation and Termination
Elongation is controlled by:
pause sites, where RNA Pol will hesitate
anti-termination, which proceeds past the normal
termination point
positive transcription elongation factor (P-TEF) and
negative transcription elongation factor (N-TEF)
Termination
begins by stopping RNA Pol; the eukaryotic
consensus sequence for termination is AAUAAA
Gene Regulation
Enhancers and silencers- regulatory sequences
that augment or diminish transcription, respectively
DNA looping brings enhancers into contact with
transcription factors and polymerase
Eukaryotic Gene Regulation
Response elements are enhancers that respond to
certain metabolic factors
heat shock element (HSE)
glucocorticoid response element (GRE)
metal response element (MRE)
cyclic-AMP response element (CRE)

Response elements all bind proteins (transcription

factors) that are produced under certain cell
conditions
Response Elements
Activation of transcription Via CREB and CBP

Unphosphorylated
CREB does not bind
to CREB binding
protein, and no
transcription occurs
Phosphorylation of
CREB causes binding
of CREB to CBP
Complex with basal
complex (RNA
polymerase and
GTFs) activates
transcription
Structural Motifs in DNA-Binding Proteins
Most proteins that activate or
inhibit RNA Pol II have two
functional domains:
DNA-binding domain
transcription-activation domain
DNA-Binding domains have
domains that are either:
Helix-Turn-Helix (HTH)
Zinc fingers
Basic-region leucine zipper
Helix-Turn-Helix Motif

Hydrogen bonding between amino acids and DNA

Zinc Finger Motif
Motif contains 2
cysteines and 2 His
12 amino acids later

Zinc binds to the

repeats
Basic Region Leucine Zipper Motif
Many transcription factors contain this motif, such as
CREB (Biochemical Connections, page 315)

Half of the protein composed of basic region of

conserved Lys, Arg, and His

Half contains series of Leu

Leu line up on one side, forming hydrophobic pocket

Helical Wheel Structure of Leucine Zipper
Transcription Activation Domains
acidic domains- rich in Asp and Glu. Gal4 has
domain of 49 amino acids, 11 are acidic

glutamine-rich domains- Seen in several

transcription factors. Sp1 has 2 glutamine-rich
domains, one with 39 Glu in 143 amino acids

proline-rich domains- Seen in CTF-1 (an activator). It

has 84 amino acid domain, of which 19 are Pro
Post Transcriptional RNA Modification
tRNA, rRNA, and mRNA are all modified after transcription
to give the functional form
the initial size of the RNA transcript is greater than the final
size because of the leader sequences at the 5 end and
the trailer sequences at the 3 end
the types of processing in prokaryotes can differ greatly
from that in eukaryotes, especially for mRNA
Modifications
trimming of leader and trailer sequences
addition of terminal sequences (after transcription)
modification of the structure of specific bases (particularly
in tRNA)
Posttranscriptional Modification of tRNA
Precursor
Modification of tRNA
Transfer RNA- the
precursor of several
tRNAs is can be
transcribed as one long
polynucleotide sequence
trimming, addition of
terminal sequences,
and base modification
all take place
methylation and
substitution of sulfur for
oxygen are the two most
usual types of base
modification
Modification of rRNA
Ribosomal RNA
processing of rRNA is primarily a matter of
methylation and trimming to the proper size

in prokaryotes, 3 rRNAs in one intact ribosome

in Eukaryotes, ribosomes have 80s, 60s, and 40s

subunits

base modification in both prokaryotes and eukaryotes

is primarily by methylation
Modification of mRNA
Includes the capping
of the 5 end with an
N-methylated guanine
that is bonded to the
next residue by a 5 ->
5 triphosphate.

Also, 2-O-methylation
of terminal ribose(s)
mRNA Modification
A polyadenylate tail that is usually100-200
nucleotides long, is added to the 3 end before the
mRNA leaves the nucleus
This tail protects the mRNA from nucleases and
phosphatases
Eukaryote genes frequently contain intervening base
sequences that do not appear in the final mRNA of
that gene product
Expressed DNA sequences are called exons
Intervening DNA sequences that are not expressed
are called introns
These genes are often referred to as split genes
Organization of Split Genes in Eukaryotes
The Splicing Reaction
Exons are separated
by intervening intron

When the exons are

spliced together,a
lariat forms in the
intron
Ribozymes
The first ribozymes discovered included those that
catalyze their own self-splicing
More recently, ribozymes have been discovered that
are involved in protein synthesis
Group I ribozymes
require an external guanosine
example: pre-rRNA of the protozoan Tetrahymena
(next screen)
Group II ribozymes
display a lariat mechanism similar to mRNA splicing
no requirement for an external nucleotide
Self-splicing of pre-rRNA