The process involved in gene transcription in Prokayotes is described in detail in this slides

1. Transcription of genes
• Definition of a gene
• Crick’s central dogma
• RNA polymerase
• Transcription cycle
• Transcription initiation in bacteria
• Transcription termination in bacteria

2. Transcription - Learning Outcomes
1. Provide a modern definition of a gene
2. Explain how either DNA strand can be used for gene
expression
3. List some differences and similarities between DNA replication
and transcription
4. Describe the core promoter elements in bacteria
5. Describe the 3 basic stages in transcription
6. Name key components of RNA Pol that allow the enzyme to
bind specifically to promoters to initiate transcription.
7. Explain what is meant by an intrinsic terminator
You should be able to:

3. Definition of a gene
• Classical definition: “The fundamental physical and functional
unit of heredity that carries information from one generation to
the next”
• Modern definition: “A unit of DNA that contains the information to
specify synthesis of a single polypeptide chain or functional RNA
(such as tRNA)”
• NB some textbooks differ with this definition

4. 1860s-1900s
1910s
1940s
1950s
1960s
1970s-1980s
1990s-2000s
Gene as a discrete unit of heredity
Gene as a discrete locus
Gene as a blueprint for a protein
Gene as physical molecule
Gene as transcribed code
Gene as open reading frame
“A unit of DNA that contains the
information to specify synthesis of
a single polypeptide chain or
functional RNA”
Word “gene” coined –
Wilhelm Johannsen
Gene mapping
Thomas Hunt Morgan
One Gene – One Protein
Beadle and Tatum
DNA is genetic material
Central dogma / genetic
code
DNA cloning and
sequencing
Genome and
“transcriptome” sequencing

5. The central dogma of molecular biology
• Determination of DNA structure in
1950’s led to this central model
DNA replication and decoding of
genetic information
• DNA, RNA and proteins are co-
linear – allows prediction

6. Most (but not all) types of RNA encode protein
• RNA can be classified as Informational or Functional
• INFORMATONAL
• messenger RNA (mRNA) – encodes proteins
• FUNCTIONAL
• Transfer RNA (tRNA) – translation
• Ribosomal RNA (rRNA) – translation
• Small nuclear RNA (snRNA) – pre-mRNA splicing
• Small cytoplasmic RNA (scRNA) – protein trafficking and
gene regulation
All are encoded by DNA-based genes

7. An E. coli physical (sequenced) map
Notes:
• One OR the other DNA strand acts as
template
• Genes rarely overlap
• Very little non-coding DNA in bacterial
genomes
zoom

8. Transcription
• Transcription - the production of RNA using DNA as a template
and ribonucleoside triphosphates as substrates
• RNA synthesis occurs in a 5’-3’ direction
• Primers are not required
• For every gene, only one of the two strands of DNA is used as
template

9. RNA is complementary to template strand
• If DNA was denatured and mixed with mRNA, the mRNA could
hybridise to the template strand

10. Transcription – mechanistic comparison with replication
DNA replication Transcription
Enzyme DNA polymerase RNA polymerase
Primer required? yes no
Direction of synthesis 5’-3’ 5’-3’
Template Both strands of DNA
copied
One strand of DNA
transcribed
Substrates Deoxyribonucleoside
triphosphosphates
Ribonucleoside
triphosphates
Displacement of new
strand
Not displaced Displaced
Accuracy 1 mistake in 107
additions
1 mistake in 104
additions

11. Transcription is selective
• DNA replication produces identical copies of the entire genome,
• Whereas
• DNA transcription selectively copies certain parts of the genome (ie
genes)
• Transcribed regions defined by DNA sequences that signal
initiation (PROMOTERS) and others that signal termination
(TERMINATORS)
• Not all genes are transcribed all of the time – GENE REGULATION

12. Transcription is highly controlled
• “Gene expression” is regulated by transcription factors
• Transcription factors affect the frequency of transcription of
specific genes
• Changes in transcription frequency usually correlate with
changes in protein level

13. RNA polymerase
• RNA polymerase moves along DNA unwinding DNA helix
• DNA is “melted” inside RNA polymerase, with template at active site
• Ribonucleotides are added to the 3’ end of nascent RNA
• A short “window” of RNA-DNA helix moves along DNA with RNA
polymerase

14. Multiple RNA polymerases on a gene
• A second RNA polymerase can start transcribing a gene before the
first one has finished.
A ribosomal RNA gene

15. Bacterial and eukaryotic RNA polymerases are related
Bacterial (Thermus aquaticus) Eukaryotic RNA Pol II (baker’s yeast)
• Crystal structures show overall shape and organisation
conserved – “crab claw” with active site at base of pincers
• Active site highly conserved– mechanism of RNA synthesis
conserved
• Less conservation in peripheral regions – differences in
communication/regulation
Mg2+

16. 3 basic phases in transcription
• INITIATION
• RNA Pol binds to a promoter (CLOSED COMPLEX)
• DNA is “melted” to reveal template (OPEN COMPLEX)
• RNA Pol catalyses production of small polyribonucleotide
then “escapes” the promoter
• ELONGATION
• RNA Pol unwinds DNA in front and re-anneals it behind,
dissociates RNA from DNA template, and proofreads.
• TERMINATION
• RNA Pol transcribes a terminator signal that causes
termination and dissociation of RNA Pol and RNA product.

17. Transcription starts at PROMOTERS and ends at TERMINATORS
5’ 3’
3’ 5’
Promoter
melting
Open complex
5’ 3’
3’ 5’
Binding
Closed complex
RNA
polymerase
5’ 3’
3’ 5’
Initiation
5’ 3’
3’ 5’
5’
Elongation
Transcription bubble
5’ 3’
3’ 5’
promoter
3’
5’
Termination
5’
3’
5’ 3’
e.g. mRNA
terminator

18. Bacterial RNA Pol requires SIGMA (s) to recognise promoters
• Bacterial “core” RNA Pol (5 protein subunits) is sufficient for
transcription elongation but it cannot recognise promoters
• A sixth subunit called SIGMA (s) allows RNA Pol to recognise
promoters
• Sigma + core RNA Pol = HOLOENZYME
• The 2 key roles of sigma are to:
1. To bind specifically to promoter elements
2. Stabilize melted DNA (open complex)
• Sigma dissociates during elongation

19. Sigma dissociates during elongation
5’ 3’
3’ 5’
promoter
HOLOENZYME
5’ 3’
3’ 5’
Binding
Closed complex
s
s
5’ 3’
3’ 5’
Promoter
melting
Open complex
s
5’ 3’
3’ 5’
Initiation s
5’ 3’
3’ 5’
5’
Elongation
Transcription bubble
s
dissociation

20. Bacterial promoters
• Comparison of sequence upstream from transcription start sites
revealed hexameric consensus promoter sequences centred 35bp and
10bp upstream (-35 and -10 elements)
• -10 consensus = TATAAT
• -35 consensus = TTGACA
• Some promoters have an UP element (T-rich) that strengthens the
promoter

21. Consensus sequences
• The alignment of related protein or nucleic acid sequences can reveal
which residues are best conserved
• If E. coli promoters are aligned the -10 and -35 elements are found to
be conserved and some positions are especially conserved.
• A consensus promoter sequence includes the sequences that are best
conserved – this is usually the “ideal” promoter.
• Deviation from the consensus will usually weaken a promoter

22. Transcription termination
• In bacteria most transcription termination does not require protein
factors.
• “INTRINSIC TERMINATORS” - DNA sequence shows DYAD
SYMMETRY – when transcribed the RNA can form a hairpin loop
Boxes show mutations that
disrupt the terminator

23. Transcription termination
RNA Pol transcribes through the
terminator sequence
(RNA Pol not shown)
A hairpin forms in the RNA
Hairpin reduces affinity of RNA Pol for
template DNA as an AT-rich section of
DNA is transcribed – the weak
interactions (U-A base-pairs) allow
dissociation of complex

24. Bacterial promoters and terminators

25. Test yourself
• Name 4 key differences between DNA replication and
transcription?
• Briefly name and describe the three basic phases of
transcription?
• What is meant by closed and open complexes in transcription
initiation?
• What are the two roles of sigma during bacterial transcription
initiation?
• What are the key elements that form bacterial promoters?
• What is an intrinsic terminator?