TRANSCIPTION

Central Dogma of Molecular Biology

DNA REPLICATION DNA

REVERSE
TRANSCRIPTION
TRANSCRIPTION

RNA REPLICATION RNA

TRANSLATION

Protein
GENERAL FEATURES OF
TRANSCIPTION
 Transcription General features
Synthesis of RNA in 5 3 on DNA template

Mechanism similar to DNA replication except:

Uses ribonucleotide triphosphate as precursors
Only one DNA strand used as template
No requirement for primers

Involves 3 steps:
Initiation
Elongation
Termination
Transcription General features
 Transcription General features
Template strand provides template (3 5) for making
complementary RNA strand (5 3)

Non-template strand strand that has identical (except

for uracil bases) sequence to that of RNA

Non-template/Coding
TCAAGCATT Non-template
5 AGTTCGTAA
TCAAGCATT Template 3
3 AGTTCGTAA 5
UCAAGCAUU
5 UCAAGCAUU RNA
3

Template/Non-coding strand

RNA
 Transcription General features
Transcription Unit:
Promoter
RNA-coding sequence
Terminator

Sequences are called Upstream and downstream

sequences

Upstream Downstream
+1
5 3
3 5
Promoter |-------------RNA coding sequence--------------------| Ter
PROKARYOTIC
TRANSCRIPTION
 Prokaryotic RNA Polymerase
Prokaryotic RNA polymerase is a ~ 500kD protein

Core enzyme comprises 4 subunits 2

Holoenzyme comprises 5 subunits - 2

Holoenzyme required for initiation, then factor

dissociates and core enzyme continues RNA synthesis

RNA polymerase places the complementary NTP and joins

it to the neighboring ribonucleotide by a phosphodiester
linkage

Synthesis occurs in 5 3 direction

Prokaryotic RNA Polymerase
Subunit Function
Alpha Assembly of core enzyme (helped by omega subunit)
Beta Ribonucleoside triphospohate binding site
Beta DNA template binding site
Sigma Promoter recognition
 Initiation
Promoter sequences upstream provide binding sites for
RNA polymerase holoenzyme

factor recognizes and binds to two consensus

sequences:
-35 sequence: Recognition sequence with the consensus
sequence 5-TTGACA-3
-10 sequence: TATA box/ Pribnow Box with the consensus
sequence 5-TATAAT-3
 Initiation
Holoenzyme binds loosely & reversibly to -35 sequence Closed Promoter Complex

Holoenzyme binds tightly to -10 sequence & locally unwinds

DNA Open Promoter Complex

First two nucleotides incorporated and phosphodiester bond formed Ternary Complex

Abortive initiation

Successful initiation & factor dissociates

Core enzyme leaves promoter, moving along template after RNA chain about 10nts.

Elongation ternary complex

 Elongation
Release of factor

Conformational change in RNA pol cannot bind to promoter

Escape from promoter Moves downstream

Transcription bubble - ~18nts

Continuous RNA synthesis on template strand 5-8 nts bound

RNA pol can proofread

DNA unwinding and rewinding at ends of transcription bubble

Topoisomerase relieves DNA positive and negative supercoils

Elongation continues until termination occurs

Transcription
Transcription
Transcription in realtime
 Termination

RNA polymerase should stop synthesis

RNA-DNA hybrid should be released completely

RNA molecule should be released from RNA polymerase

RNA polymerase should detach from DNA

 Termination
Two types of terminators:
Rho-independent
Rho-dependent
 Rho-independent termination
Rho-independent terminator have the following
sequences:
Inverted repeats that can cause hairpin loops in RNA after
transcription

String of adenines downstream to repeats that produce weak

A=U bonds in the RNA-DNA hybrid
Rho-independent termination
RNA polymerase transcribes inverted repeats

Repeats on RNA fold into hairpin loop

Loop causes RNA polymerase to pause

RNA-DNA hybrid region now contains weak A=U bonds that break

RNA transcript is released

Transcription is terminated
Rho-independent termination
 Rho-dependent termination
Rho-dependent terminator have the following sequences:
Upstream to terminator sequence is a sequence that codes for
RNA that cannot form any secondary structure and provide
binding site for Rho rut site rich in C
Terminator sequence that causes RNA polymerase to pause

Rho factor
~ 75kD protein
Hexameric protein
ATP-dependent helicase
Each subunit has RNA-binding domain and ATP hydrolysis domain
 Rho-dependent termination
RNA polymerase transcribes ter-upstream sequence producing unstructured RNA

Rho protein binds to this unstructured part of RNA

Bound Rho protein moves toward the 3 end of the RNA molecule

RNA polymerases reaches and transcribes terminator sequence

RNA polymerase pauses

Rho protein catches up with stalled RNA polymerase

Helicase activity of Rho unwinds RNA-DNA hybrid

RNA released and transcription terminated

Rho-dependent termination
 Antitermination
Anti termination proteins enable RNA polymerases to
read past a specific terminator and continue
transcription of genes beyond the terminator sequence

Antiterminator proteins act on sites upstream of the

terminator sites

Antitermination used as control mechanism in phage

regulatory circuits and bacterial operons
EUKARYOTIC
TRANSCRIPTION
 Eukaryotic RNA Polymerases

Three RNA polymerases I,

II, and III

All three are complexes with

more than 10 subunits

Eukaryotic RNA pols require

various transcription factors
to initiate transcription

Each RNA polymerase

transcribes a specific sets
of genes
 Eukaryotic RNA Polymerases
Enzyme Transcribes Location

RNA polymerase I Large rRNA (All except 5S rRNA) Nucleolus

Pre-mRNA, few snRNA, snoRNA, few

RNA Polymerase II Nucleus
miRNA

RNA Polymerase III tRNA, 5S rRNA, other small RNA Nucleus

A poorly understood fourth RNA pol called RNA Polymerase IV has

been found in plants that transcribe siRNA

Pol V seems to transcribe intergenic DNA, these transcripts

providing a scaffold for the siRNAs
 Eukaryotic Promoters
Two regions:
Core promoter elements (basic transcription initiation)
Regulatory promoter elements (determine efficacy of promoter)

Core promoter:
Typically not more than 50 bp upstream of +1
Ini (initiator element) at +1
TATA box or element (Goldberg-Hogness box) at -30 (TATAAA)

Regulatory promoter
50-200 bp upstream of +1
CAAT box at -75
GC box at -90
 Eukaryotic Promoters
Protein coding genes have a combination of core promoter elements and regulatory
promoter elements
 Eukaryotic Promoters
Different promoter for different RNA polymerases
 Eukaryotic Promoters
Transcription factors (TFs)
General TFs (GTFs) bind at the core promoter
GTFs are named with roman numerals after the RNA pol they
work with and lettered in the order of their discovery (example:
TFIIB is the fourth discovered TF acting with RNA pol II)
Activators bind to regulaotyr promoter elements and aid in
regulation of trnascription

Enhancers
Sequences that regulate transcription at a distance
Can be present upstream and downstream, usually upstream
Can be present thousands of bp away from the gene they control
 Initiation
TFIID (composed of TBP and TAFs) recognizes and binds to TATA box Initial
committed complex

TFIID-TATA box complex forms a binding site for sequential addition of other TFs

Binding of TFIIA followed by TFIIB

RNA pol II recruited and TFIIF binds minimal

transcription initiation complex

TFIIE and TFIIH bind complete transcription initiation

complex or pre-initiation complex (PIC)

TFIIH has helicase activity that unwinds DNA locally (11-15bp)

 Elongation
Abortive initiation

After 30 bases on RNA have been synthesized, promoter

escape occurs

Most TFs are left behind - reinitiate by another RNA pol molecule

~8 bases remain as RNA-DNA hybrid within the transcription bubble

5 end modified by addition of 7-methyl guanosine (7-MG) cap

As RNA pol moves, histones are removed and replaced by

FACT (facilitates chromatin transcription)
Transcription in realtime
Eukaryotic Transcription
 Termination
RNA pol I:
Similar to rho-dependent termination
Termination factor binds to DNA sequence downstream of
terminator

RNA pol III:

Similar to rho-independent termination
Terminator sequence has string of A, producing A=U bond
Dissociation happens without the requirement of hairpin
structure
 Termination
No specifc termination site for RNA pol II

Transcription continues beyond coding sequence

Pre-mRNA cleaved at a specific consensus sequence on RNA

Rat1 attaches to 5 end generated by cleaving, while RNA pol

continues transcription

5 3 exonuclease activity degrades RNA as it moves toward 3 end

When Rat1 reaches RNA pol II transcription is terminated

Termination