1. Gene expression. Transcription

2. Evolution of the concept «gene»
W. Johannsen introduced the word "gene“ (1909)
Genes are physical and functional units of heredity (in the early 20th century)
A gene is a locus on a chromosome (Т. Morgan, 1910)
the "one gene, one enzyme" hypothesis (G. W. Beadle and E. L. Tatum, 1945)
A gene is a sequence of nucleotides that encodes a specific functional product (a protein or RNA molecule).

3. Genes inducible genes Structural genes regulatory genes
constitutively expressed genes
Luxury genes
Housekeeping genes

4. Gene expression

5. Transcription Transcription is the process of making an RNA copy of a DNA molecule.
Conditions
DNA template
NTPs
Enzymes
Energy (ATP) Mg
Principles
a template is required
Complementary
Antiparallel

6. A transcripton is a transcription unit
A transcripton is a transcription unit. A promoter is a DNA sequence to which RNA polymerase binds to initiate transcription. A terminator is a DNA sequence that marks the end of transcription.

7. Structure of a prokaryotic transcripton
Promoter
Coding region
Terminator 5’ 3’
3’-untranslated region
5’-untranslated region

8. Structure of a prokaryotic promoter
-35
-10 +1 5’ 3’ 3’ 5’
TTGACA
(-35 element)
ТАТААТ
(-10 element or Pribnow box)
CAT
(Start point)

9. Prokaryotic RNA polymerase
1. RNA polymerase has some subunits:α, α, β, β’, ω, σ.
2 α assemble of RNAP
β forms phosphodiester bonds
β’ (non-sequence-specific interactions with DNA )
ω assembles RNAP and stabilizes it
σ recognizes promoters
ααββ’ω – core enzyme
Core enzyme + σ factor = holoenzyme
There are some different σ factors in prokaryotic cells.
2. The enzyme catalyzes the reaction
(NMP)n + NTP → (NMP)n+1 + PP
3. RNA polymerase synthesizes RNA only in the direction 5’→3’.
4. RNA polymerase doesn’t require a primer (it’s able to initiate RNA synthesis de novo).
Catalytic (active) site
of RNA polymerase

10. Initiation of transcription (prokaryotic cells)
Binding of RNA polymerase (holoenzyme) to the promoter in DNA (closed complex)
Formation of an open complex (the DNA is unwound)
The RNA polymerase (holoenzyme) transcribes the first 8-10 nucleotides.
σ subunit dissociates from the holoenzyme.

11. Elongation of transcription (prokaryotic cells)
The RNA polymerase (core enzyme) moves down the DNA template, opens DNA and adds nucleotides to the growing RNA chain.

12. Termination (prokaryotic cells)
Termination is the final step of transcription. It results in the release of the newly synthesized RNA from the elongation complex.
Termination
ρ-independent
ρ-dependent

13. ρ-dependent termination
The rho factor is thought to bind to the RNA chain and slide along the strand towards the open complex bubble. When the factor catches the polymerase, it causes the termination of transcription.
Rho factor has a helicase and ATPase activities.

14. Structure of a eukaryotic transcripton
exons 5’ 3’
introns
terminator
promoter
5’-untranslated region
3’-untranslated region

15. Transcription (eukaryotic cells)
RNA polymerases:
RNA pol I synthesizes a pre-rRNA 45S, which matures into 5.8 S, 18 S, 28 S rRNAs.
RNA pol II synthesizes precursors of mRNAs and most snRNAs and microRNAs.
RNA pol III synthesizes tRNAs, 5 S rRNA and some small RNAs .
Mitochondrial RNA pol

16. Structure of a eukaryotic promoter (for RNA pol II)
-100/-300
-60/-80
-25 +1 5’ 3’ 5’ 3’
GC box
СААТ box
ТАТААА
(ТАТА box, Goldberg –Hogness box)
CAT
(PyAPy)
(start point)

17. Transcription factors (for RNA pol II)
ТВР binds specifically to ТАТА box
TAF – TBP-associated factors
TF II A stabilizes binding between TFIID and promoters; acts as a coactivator for some transcriptional activators
TF II B functions as a linker between TFIID and RNA pol II
TF II F binds to RNA pol II
TF II H has helicase and ATPase activities and aids in creation of the transcription bubble. It phosphorylates the polymerase II (kinase activity)
TF II E is required for TH II H binding and transcription stimulation
TF II J (the function is unknown)
TF II D

18. RNA processing
RNA processing is a complex of post-transcriptional modifications of the RNA molecule (RNA maturation).

19. Steps of mRNA processing (eukaryotic cells)
5′ capping
2. 3' polyadenylation
3. Splicing

20. 5′ capping is addition of a 5’ 7-methyl guanosine.
Further methylation of the first 2 ribose sugars of the 5′ end of the mRNA is possible.
A guanine nucleotide connected to the mRNA via an unusual 5′ to 5′ triphosphate linkage.
Capping happens before transcription is finished = co-transcriptionally
Enzymes: a guanylyl transferase, methyl transferases

21. Types of caps
Cap 0 – the cap is methylated at the N-7 position of guanine
Cap 1 – guanine and the first nucleotide are methylated
Cap 2 – guanine, the first and second nucleotides are methylated.

22. Functions Cap: polyA-tail:
Protects the growing RNA chain from degradation by exonucleases.
Is involved in export of the mRNA from the nucleus
Is recognized by the translation machinery.
Is involved in 5′ proximal intron excision.
polyA-tail:
Protects the RNA chain from degradation
Aids in initiation of translation
May be involved in splicing

23. Splicing (R.J. Roberts and P.A. Sharp, 1977)
Splicing is the mechanism by which introns are removed and exons are joined.
Introns (intragenic regions ) aren’t expressed in proteins.
Exons – (expressed regions) are retained in the mature mRNA.
Kinds of introns:
Group I introns are in rRNA genes of lower eukaryotes (self-splicing)
Group II introns are in mitochondrial genes (self-splicing)
Group III introns are in nuclear protein-coding genes (removed by spliceosomes)

24. Structure of a mature mRNA (eukaryotic cells)
Protein-coding segment
Polyadenylation signal 5 3 G P P P
AAUAAA
AAA …
AAA
Start codon
Stop codon 5
Cap 5
UTR 3
UTR
Poly-A tail
UTR – untranslated region

25. tRNA processing (eukaryotic cells)

26. rRNA processing (eukaryotic cells)

27. rRNA processing (prokaryotic cells)
tRNA
16S
23S 5S
Pre-RNA
RNases:
III
III III
III
P P
16S
23S 5S
rRNAs:

28. tRNA processing (prokaryotic cells)