Gene Expression in prokaryotic cells

1. Gene expression
Mohammad Ridhuan Mohd Ali
Bacteriology Unit
Infectious Disease Research Centre (IDRC)
Institute for Medical Research (IMR), Malaysia

2. Central dogma
• first proposed in 1958
by Francis Crick
• the process by which
the instructions in
DNA are converted
into a functional
product
• DNA->RNA->protein

3. Regulation of transcription
• Transcription factor (TF)
• protein that regulate gene expression
• binding to specific sequences that are associated with a transcription
initiation site

4. Regulation of transcription
• Transcription factor (TF)
• protein that regulate gene expression
• binding to specific sequences that are associated with a transcription
initiation site
Activator

5. Regulation of transcription
• Transcription factor (TF)
• protein that regulate gene expression
• binding to specific sequences that are associated with a transcription
initiation site
Activator Repressor

6. Regulation of transcription
• Transcription factor (TF)
• protein that regulate gene expression
• binding to specific sequences that are associated with a transcription
initiation site
Activator Repressor
Enhancer

7. Regulation of transcription
• Transcription factor (TF)
• protein that regulate gene expression
• binding to specific sequences that are associated with a transcription
initiation site
Activator Repressor
Enhancer Silencer

8. Regulation of transcription
• Transcription factor (TF)
• protein that regulate gene expression
• binding to specific sequences that are associated with a transcription
initiation site
Activator Repressor
Enhancer Silencer

9. Regulation of transcription
• Histone acetylation
Condensed chromatin
Relaxed
chromatin
Eslaminejad et al 2013

10. Regulation of transcription
• Histone acetylation
Relaxed
chromatin
Condensed chromatin
http://helicase.pbworks.com/

11. Transcription in eukaryotes
• Involves three different enzymes:
• RNA pol. I
• located in the nucleolus
• catalysed the synthesis of all rRNAs except the small 5S rRNA.
• RNA pol. II
• transcribes nuclear genes that encode proteins and other proteins
• specifying hnRNAs (heterogenous nuclear RNAs).
• hnRNAs also known as pre-mRNA
• RNA pol. III – catalysed the synthesis of tRNAs, 5S rRNAs and snRNAs.

12. Transcription in eukaryotes
• All three RNA polymerase require the assistance of transcription factors (TF) to initiate the synthesis
of RNA chains.
• exhibit different sensitivities to α-amanitin, metabolic poison from mushroom Amanita phalloides
• α-amanitin can be used to determine which RNA polymerase catalyzes the transcription of a
particular gene.

13. Transcription in eukaryotes
• Initiation
• Eukaryotic RNA pol. requires transcription factors (TFs) to
initiate the RNA synthesis.
• TFs must bind to the promoter region in DNA and form a
complex, before RNA pol will bind and initiate transcription.
• Promoters recognized by RNA pol II has a short conserved
element, located upstream the transcription start point.

14. Transcription in eukaryotes

15. Transcription in eukaryotes
• Initiation
• begins ways before the transcription start point
• Requires an orchestra of RNA polymerase and transcription factors (TFs)

16. Transcription in eukaryotes
• Elongation
• Once the RNA polymerases has been released from the initiation complex,
the elongation process is the same as in prokaryotes.
• 7-methyl guanosine (MG) caps are added at the 5’ ends of pre-mRNA, shortly
after the elongation process begins (about 30 nucleotides long).
• 7-MG caps are recognized by factors involved initiation of translation
• Protect the growing RNA chains from nucleases.

17. • Termination
• The 3’-ends of the RNA transcripts
are produced by endonuclolytic
cleavage, rather than termination
of transcription.
• The actual termination occurs 1k-
2k nucleotides, downstream from
the 3’ end of mature transcripts
• Between AAUAAA andG-U rich
sequence
Transcription in eukaryotes

18. Transcription in eukaryotes
• After cleavage, the enzyme poly(A) polymerase adds poly(A) tails
about 200 nucleotides to the 3’ ends (polyadenylation).
• Poly A tails
• enhance the stability of the mRNAs
• play important role in their transport from nucleus to the cytoplasm.

19. Pre-mRNA in eukaryotes

20. Transcription in prokaryotes
• Initiation
• at specific promoters
• Prokaryote RNA polymerase
• Has 5 subunits of polypeptides
• ß’ – largest subunit
• ß – second largest
• αI – NTD assembly of RNAP
• αII – CTD bind to promoter
• Ω – assist, stabilise RNAP to bind to the promoter

21. Transcription in prokaryotes
• Initiation
• at specific promoters
• Prokaryote RNA polymerase
• Has 5 subunits of polypeptides
• ß’ – largest subunit
• ß – second largest
• αI – NTD assembly of RNAP
• αII – CTD bind to promoter
• ω – assist, stabilise RNAP to bind to the promoter
• Require σ factor to initiate the transcription

22. Transcription in prokaryotes
• Promoter sites
• The sequence vary from gene to gene, but some
are highly conserved; Consensus sequence
• The -10 consensus sequence in the non-
template strand is 5’-TATAAT-3’ (Pribnow box);
this A:T rich region facilitates the localized
unwinding of the DNA.
• The -35 consensus sequence is 5’-TTGACA-3’
(also called the recognition sequence, subunit
initially recognize and binds to this sequence).
• Distance between the two sequences is 15 -20
bp

23. Transcription in prokaryotes
• Elongation

24. Transcription in prokaryotes
• Termination
• Happen in two ways
• Rho independent

25. Transcription in prokaryotes
• Termination
• Happen in two ways
• Rho independent
• Rho dependent

26. Transcription in prokaryotes
• Termination
• Happen in two ways
• Rho independent
• Rho dependent
• Rho hexameric protein

27. Transcription in prokaryotes
• Termination
• Happen in two ways
• Rho independent
• Rho dependent
• Rho hexameric protein
• Rho Utilisation Site (Rut site) (Cystine-rich)

28. Transcription in prokaryotes
• Termination
• Happen in two ways
• Rho independent
• Rho dependent
• Rho hexameric protein
• Rho Utilisation Site (Rut site) (Cystine-rich)
• In prokaryotes, translation and degradation of an mRNA often begin before
termination
• mRNA molecules are synthesized, translated and degraded in the 5’ to 3’
direction,
• all three processes can occur simultaneously on the same RNA molecule.

29. Transcription in prokaryotes
• Termination
• Happen in two ways
• Rho independent
• Rho dependent
• Rho hexameric protein
• Rho Utilisation Site (Rut site) (Cystine-rich)
• In prokaryotes, translation and degradation of an mRNA often begin before
termination
• mRNA molecules are synthesized, translated and degraded in the 5’ to 3’
direction,
• all three processes can occur simultaneously on the same RNA molecule.

30. monocistronic
polycistronic

32. mRNA vaccine
Element Description Position
cap A modified 5’-cap1 structure (m7G+m3'-5'-ppp-5'-Am) 1-2
5’-UTR 5´-untranslated region derived from human alpha-globin RNA with an optimized Kozak
sequence
3-54
sig S glycoprotein signal peptide (extended leader sequence), which guides translocation of
the nascent polypeptide chain into the endoplasmic reticulum.
55-102
S protein_mut Codon-optimized sequence encoding full-length SARS-CoV-2 spike (S) glycoprotein
containing mutations K986P and V987P to ensure the S glycoprotein remains in an
antigenically optimal pre-fusion conformation; stop codons: 3874-3879 (underlined)
103-3879
3’-UTR The 3´ untranslated region comprises two sequence elements derived from the amino-
terminal enhancer of split (AES) mRNA and the mitochondrial encoded 12S ribosomal
RNA to confer RNA stability and high total protein expression.
3880-4174
poly(A) A 110-nucleotide poly(A)-tail consisting of a stretch of 30 adenosine residues, followed by
a 10-nucleotide linker sequence and another 70 adenosine residues.
4175-4284

33. Thank you

34. 1% of DNA are encoded for protein
99% are non-coding DNA / junk

35. Non-coding DNA / junk
Satellite DNA
Telomeres
Introns
Non-coding RNA
Gene regulatory sequences