Transcription

• Definition
• Terms used in
transcription
• Stages of transcription in
prokaryotes
• Transcription in
eukaryotes as compared
to prokaryotes
 Transcription

• Transcription is the first stage of gene expression, in

which, by using DNA as a template, an RNA copy
(called transcript) of the DNA sequence of a gene is
produced.
• DNA RNA Protein = gene expression
• Or the central dogma of molecular biology
 Terms used

• Gene or transcriptional unit

• Upstream and downstream directions
• Template strand = antisense (-), non-coding
strand,
• Non-template strand = sense(+),coding strand
Transcription of the DNA
template strand produces
an RNA transcript with the
same sequence as the
DNA nontemplate strand,
except that U replaces T.
Chemical reaction involved in the synthesis
of RNA on a DNA template strand
 Requirements for transcription
• Substrates for RNA synthesis: NTPs
• NTPs function as building blocks & energy sources
• The enzyme needed is RNA polymerase (RP).
• As with DNA synthesis, the transcript is synthesized in
the 5’→3’ direction.
• The template strand of DNA runs in opposite.
• RP lacks proofreading activity and does not need a primer
• Protein factors may be needed during initiation,
elongation and termination
 Transcription in prokaryotes

• Three phases: initiation, elongation and

termination.
• One RNA polymerase is responsible for
RNA synthesis.
• This enzyme is made up of five core
subunits, α2ββ́ + sigma factor, σ
• The holoenzyme = (α2ββ́ + σ)
Prokaryotic RNA polymerase

• The four subunits

(α2ββ́) have known
roles in transcription
but:
• The function of the
fifth subunit of the
core enzyme () is
unknown
• Sigma is needed for
initiation only.
How a gene looks like?
 Initiation
• In E. coli, two consensus sequences in the promoter:
-10 sequence (TATAAT or Pribnow box) and -35
sequence (TTGACA box) sequence exist.
• These sequences are important for accurate
initiation of transcription
• Sequences like these (found on the same template
DNA) which influence transcription are called cis-
acting elements.
• RP (helped by σ factor) recognizes these sequences
and therefore initiates transcription correctly.
Structure of the prokaryotic
promoter region
 Initiation
• The σ-subunit of RNA polymerase holo-enzyme
recognizes and binds -35 box→
• a closed (DNA remains as a double helix )
promoter complex is formed →
• DNA double helix partially dissociate at the -10
box to give an open promoter complex →
• σsubunit dissociates leaving core enzyme →
• the first two nucleotides bind to DNA and the first
phospho-diester bond forms
Formation of the phospho-diester bond
 Elongation
• The RNA polymerase moves along the DNA
unwinding the double helix as it progresses.
• RNA molecule grows by the addition of NTPs that
are paired with DNA template.
• In most cases, a leader sequence is transcribed
before the coding sequence of a gene is reached.
• A trailer sequence is also added at the end.
• Only a small portion of the double helix (about 17
bp) is unwound at any one time.
• RNA that is made is released from the template
DNA to allow the DNA double helix to reform.
Elongation complex
 Termination
• Termination involves release of the RNA
transcript and the core enzyme and initiation of
another round of transcription.
• Two strategies are used by prokaryotes for
termination of transcription.
• Rho-dependent, and Rho-independent
termination of transcription
• Rho-independent termination:
• Termination takes place at specific points after
the end of the coding sequence.
• In E. coli, termination occurs at palindromes,
such that a stem-loop structure of RNA will form
after transcription.
• A run of 5-10 A’s usually follow palindromes
such that weak A:U base pairs be formed with
the newly synthesized RNA.
• Rho-dependent termination:
• RNA polymerase pauses after the stem-
loop, when the run of As is absent, Rho
protein binds RNA and disrupts base
pairing between the template and the
transcript
 Transcription in eukaryotes
• Similar to prokaryotes but:
• is more complex in initiation,
• complexity of the chromatin structure
• termination does not involve stem-loop structure
and
• is carried out by three RNA polymerases.
Eukaryotic gene promoter
consensus sequences
 RNA polymerase II
• Transcribes genes that encode proteins
• (Produces mRNA)
• Promoter:
– TATA box: TATA(A/T)A(A/T), position -25
– CCAAAT box at -70
– In addition to GC-rich boxes in between
– All these sequences are very vital for
interaction with numerous transcriptional
factors and with RNA polymerase
– Transcription factors: TFⅡD, TFⅡA, TFⅡB,
TFⅡF, TFⅡE, TFⅡH, TFⅡJ bind to the DNA around
the TATA box and form a platform for RNA
polymerase Ⅱ to bind.
– TF bind in a specific order .
– Other regulatory sequences
(1) enhancers, located far away from the
gene, increase the rate of transcription,
enhancer can be located upstream or down
stream (2) silencers, inhibit
transcription
• A – Eukaryotic general
transcription factors bind to
consensus sequences in the
promoter.
• These factors are needed for the
formation of the initiation
complex and for recruitment of
the RNA polymerase

• B- Enhancer-binding
transcription factors can also
stimulate the activity of the
polymerase
 Termination
• Termination: destabilizes transcription
complex by dissociation of transcription
factors.
• Initiation by RNA polymerase I and III:
similar to polymerase II
 RNA polymerase I
• Transcribes 18S, 28S and 5.8S rRNA
genes.
• Promoter sequences: (1) core element,
essential for transcription to occur; (2)
upstream control element, stimulate
transcription.
• Terminal signal: a 18bp consensus
sequence present about 600bp
downstream from the end of the gene.
 RNA polymerase III
• Transcribe tRNA and 5S rRNA genes.
• Promoter sequences: lie in transcribed
sequences (+100 or so), called internal
control region (ICRs).
 Post-transcriptional modifications
RNA Processing
• Changes that happen on the primary transcript are
called post-transcriptional modifications.
• These include: base modifications (eg by
methylation) and addition or removal of certain
nucleotides.
• Modifications that happen on the primary transcript
of mRNA (called hnRNA) are the most extensive
ones and differentiate between prokaryote and
eukaryotes
tRNA Processing
rRNA Processing
mRNA Processing
Splicing