Chapter Eleven
Transcription of the Genetic Code:
The Biosynthesis of RNA
Chapter 11
Transcription
• Overview of Transcription
• synthesized on a DNA template, catalyzed by DNAdependent RNA polymerase
• ATP, GTP, CTP, and UTP are required, as is Mg2+
• no RNA primer is required
• 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 RNA chain is synthesized in the 5’ -> 3’ direction
• the DNA template is unchanged
Transcription in Prokaryotes
• E. coli RNA Polymerase:
–
–
–
–
–
molecular weight about 500,000
four different types of subunits: ,  , ’, and s
the core enzyme is 2’
the holoenzyme is 2’s
the role of the s subunit is recognition of the promoter locus;
the s 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;
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
How does RNA polymerase know
where to begin transcription?
• Polymerase moves along template strand from
3’-5’ and RNA is formed from 5’-3’
• Binding site for polymerase is upstream of
start of transcription – away from 5’ of coding
strand
• Promoter sequence is based on coding strand
and RNA polymerase is binding to template
strand
How does RNA polymerase know
where to begin transcription?
• Promoter are upstream towards 5’ of coding
and 3’ template strand
• The first base to be incorporated is at position
+ 1 = transcription start site
• All nucleotides upstream from this start site
are given –ve numbers
• The first promoter element is about 10 bases
upstream is – 10 region or pribnow box
How does RNA polymerase know
where to begin transcription?
• After PB 16-18 bases are variable
• Next promoter element is about 35 bases
upstream of TSS = -35 region or -35 element
• Area from -35 element to TSS = core promoter
• Upstream of core element is UP element =
enhances binding of RNA polymerase
• Region from UP element to TSS = extended
promoter
• BASE SEQUENCE of promoter is A and T
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 Transcription
Chain Elongation (Cont’d)
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 Termination
• Two types of termination mechanisms:
• intrinsic termination- controlled by specific
sequences, termination sites
• Termination sites characterized by two inverted
repeats
Chain Termination
• 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 s factors
– enhancers
– operons
– transcription attenuation
Alternative s factors
• Viruses and bacteria
exert control over which
genes are expressed by
producing different ssubunits that direct the
RNA polymerase to
different genes.
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
Example of Operon system
• -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
Transcription in Eukaryotes is complex
•
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 in
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 .
Pol II promoters
• Variety of upstream elements – activators and
silencers
- GC box (-40) – Consensus sequence –
GGGCGG
- CAAT box (extending to – 110) – Consensus
sequence - GGCCAATCT
Pol II promoters
• Second element found at -25 = TATA box has
consensus sequence of TATAA (T/A)
• Transcription start site at position + 1
surrounded by a sequence called initiator
element (Inr)
• Inititator and TATA box = core promoter
• Fourth element – downstream regulator rare
Initiation of Transcription
• Transcription factor Any protein regulator of
transcription that is not
a subunit of Pol II
• Initiation begins by
forming the
preinitiation complex Transcription control is
based here
Transcription Order of Events
• The phosphorylated
Pol II synthesizes
RNA
• 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
– positive transcription elongation factor (P-TEF)
and negative transcription elongation factor (NTEF)
• 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
Response elements
• 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)
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 –
after every 12 amino
acids
• Zinc binds to the
repeats
Basic Region Leucine Zipper Motif
• Many transcription
factors contain this motif
- CREB
• Half of the protein
composed of basic region
of conserved Lys, Arg, and
His
• Half contains series of Leu
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
• Modifications
– trimming of leader and trailer sequences
– addition of terminal sequences (after transcription)
– modification of specific bases (particularly in tRNA)
Modification of tRNA
• Transfer RNA– trimming, addition of
terminal sequences, and
base modification - 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 - methylation and trimming to
the proper size
Modification of mRNA
• Capping of the 5’ end with an N-methylated
guanine
• A polyadenylate “tail” that is usually100-200
nucleotides long, is added to the 3’ end before
the mRNA leaves the nucleus
Organization of Split Genes in Eukaryotes
Modification of mRNA
– 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
The Splicing Reaction
• Exons are separated
by intervening
intron
• When the exons are
spliced together lariat forms in the
intron
Ribozymes
• The first ribozymes
discovered included
those that catalyze their
own self-splicing
• ribozymes have been
discovered that are
involved in protein
synthesis
• Group I and II
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