Genetic expression transcription

1. GENETIC EXPRESSION:
TRANSCRIPTION
JOSHUA P. DESAMPARADO, RMT

2. Transcription
 is the process of making an RNA copy of a gene’s DNA
sequence. This copy, called messenger RNA (mRNA), carries
the gene’s protein information encoded in DNA.
 mRNA moves from the cell nucleus to the cell cytoplasm
(watery interior), where it is used for synthesizing the encoded
protein.

3. General features of RNA synthesis
 Template strand
Directs the synthesis of the RNA
Also called antisense strand
Catalyzed by DNA-dependent RNA polymerase
Adenosine triphosphate (ATP), Guanosine-5’-triphosphate (GTP),
Cytidine triphosphate (CPT) and Uridine-5′-triphosphate (UTP)
are required
Directs the synthesis of the RNA

4. General features of RNA synthesis
 Coding strand
Sequence is the same as the RNA produced with the exception
of U replacing T
Also called sense strand
RNA polymerase moves along the template in 3’ -> 5’ direaction

5. General features of RNA synthesis
Promoters
 Are DNA sequences that provide signal for RNA polymerase and
they are where RNA polymerase binds
 Binding site for polymerase is upstream of the transcription start
site. 5’ – 3’ side of the template strand
 Given based on the coding strand

6. Process of Transcription
Initiation
 Begins when RNA polymerase binds to the promoter and forms the close
complex
 Has four steps:
1. Formation of a closed promoter complex
2. Conversion of the closed promoter complex to an open promoter
complex
3. Polymerizing the first few nucleotides while the polymerase remains at
the promoter
4. promoter clearance, in which the transcript becomes long enough to
form a stable hybrid with the template strand.

7. Process of Transcription
Elongation
 RNA polymerase directs the sequential binding of ribonucleotides
to the growing RNA chain in the 5’ 3’ direction, while the RNA
polymerase and transcription bubble move along the template
DNA in 3’ 5’ direction
 As the RNA polymerase moves along the template DNA, the
transcription bubble also moves with it.

8. Process of Transcription
Termination
 Involves specific sequences downstream of the actual gene for the
RNA to be transcribed
 Two mechanisms:
 Intrinsic termination – termination sites can signal the termination of the
transcription
 rho (p) – dependent termination – involves an inverted repeat, binds to RNA
and chases the polymerase

9. Regulation of prokaryotic transcription
Has 7 major categories:
1. Alternative Sigma (σ) Factors
2. RNA polymerase switching
3. Antitermination
4. Enhancer
5. Operon
6. Transcription attenuation
7. riboswitch

10. Regulation of prokaryotic transcription
RNA polymerase switching
 Specific gene encodes a new RNA polymerase with absolute
specificity for the gene

11. Regulation of prokaryotic transcription
Has 7 major categories:
1. Alternative Sigma (σ) Factors
 Binds the catalytic core RNA polymerase to confer promoter selectivity on the
holoenzyme
2. RNA polymerase switching
 Between inactivated and activated states By translocating back and forth along the
DNA and the RNA
3. Antitermination
 are proteins that prevent termination of transcription at specific site
4. Enhancer
 Elements that stimulate transcription and are cis-acting DNA elements that are not
strictly part of the promoter

12. Regulation of prokaryotic transcription
Has 7 major categories:
5. Operon
 a cluster of genes that are transcribed together to give a single messenger
RNA (mRNA) molecule
6. Transcription attenuation
 regulatory mechanism that causes premature termination of transcription
under certain conditions, thereby preventing the expression of the mRNA
required for expression of the corresponding gene products
7. Riboswitches
 A region usually in the 5’-unstranalted region of an mRNA. Contains two
molecules: an aptamer and an expression platform

13. Transcription in eukaryotes
Eukaryotic RNA polymerase
 Needs three RNA polymerases with different activities and each one transcribes a
different set of genes and recognizes a different set of promoters
 RNA polymerase I – precursor of most ribosomal RNA
 RNA polymerase II – synthesizes mRNA precursors
 RNA polymerase III – synthesizes the tRNA, precursor of 5S rRNA and some small RNA
molecules involved in mRNA processing and protein transport
Promoter structure
 RNA polymerase II (class II promoters): core and proximal
 Core promoter can attract general transcription factors and RNA polymerase and
determents the site and direction of the transcription. Contains initiator box and TATA
box
RNA polymerase I and III-Directed Transcription
 Rely on a distinct set of proteins to initiate transcription. Although both RNA
polymerases I and III share several identical core enzyme subunits with RNA polymerase
II, they recognize very different promoter sequences and have unique general
transcription factors.

14. Transcription in eukaryotes
Order of events
 Transcription factors – needed to bind to their promoters
 General transcription factors – combine with RNA polymerase to form
a pre-initiation complex that is competent to initiate transcription
 Gene specific transcription factors (activators) – can either stimulate or
inhibit the transcription by RNA polymerase II and they have two
functional domains:
DNA-binding domain: Zinc-containing molecules, homeodomains and
bZIP and bHLH motifs
Activation domain

15. Regulation Transcription in eukaryotes
 Most eukaryotic promoters and regulatory elements are organized
into precise architecture within extensive nucleosome position sites
 To activate transcription, the transcription machinery has to
compete with histones for DNA sites that are blocked or obscured
by nucleosomes on the promoter
 It is also possible that the wrapping of nucleosomal DNA over the
surface of the histone octamer may be sterically incompatible with
the assembly of a stable transcription preinitiation complex

16. Transcription elongation through the
nucleosomal barrier
 DNA remains packaged in nucleosomes in the coding region of
transcribed genes.
 RNA pol II encounters a nucleosome approx. every 200 bp. It
overcomes this through the existence of two distich mechanisms for
the progression of RNA polymerases through chromatin:
nucleosome mobilization and H2A-H2B dimer depletion

17. Transcription elongation through the
nucleosomal barrier
Nucleosome mobilization
 Nucleosomes are translocated without release of the core octamer
into solution. Means, DNA is displaced from the nucleosomes and
the nucleosomes are transferred to a region of DNA already
transcribe by RNA pol II
 Facilitated by elongation factor FACT (Facilitates chromatin
transcription) – promotes transcription-dependent nucleosome
alterations.

18. Transcription elongation through the
nucleosomal barrier
H2A-H2B dimer Removal
 Disrupts nucleosomes on active RNA pol II transcribe genes.
 Requires the following in vitro: FACT, elongator and TFIIS

19. Posttranscriptional Modification of mRNA
 Eukaryotic genes frequently contain intervening sequences that do
not appear in the final mRNA for that gene produced.
 Exons: DNA sequences that are expressed
 Introns: Intervening sequences

20. Posttranscriptional Modification of mRNA
The Cap at the 5’ end of eukaryotic mRNA
 a guanylate residue that is methylated at the N-7 position.
 The presence of the cap can protect the mRNA from exonucleases
degradation.
 essential for the efficient elongation and termination of
transcription as well as the subsequent processing of the mRNA
 Functions as a binding site for proteins that export the mRNA from
the nucleus to the cytoplasm and for directing the initiation of protein
synthesis from the mRNA

21. Posttranscriptional Modification of mRNA
Capping and polyadenylation process
 5’ capping occur after the growing RNA emerges from RNA polymerase II
 3 steps
1. RNA 5’ triphosphate catalyzes the removal of one phosphate from the
triphosphate at the 5’ end of the mRNA
2. Guanyl transferase attaches a molecule of guanosine monophosphate (GMP)
to this end
3. Transferred guanine is methylated by a guanine-7-methyltransferase
 Polyadenylation of the 3’ end of eukaryotic mRNA begins with an initial
cleavage of the mRNA. Usually occurred after a CA nucleotide pair that
lies somewhere between a conserved AAUAAA hexamer sequence and a
U or GU-rich region further downstream.

22. Posttranscriptional Modification of mRNA
Capping and polyadenylation process
 After cleavage, a tail of approximately 200 adenosines is added by
poly (A) polymerase.
 Coupled to each other and to transcription by RNA pol II

23. Posttranscriptional Modification of mRNA
RNA splicing
 Mature transcript for many genes is encoded in a discontinuous
manner in a series of discrete exons.
 mRNA, tRNA, rRNA all contains introns that must be removed from
precursor RNA to produce functional molecules.
 They are catalyzed by the following:
 tRNA precursors - protein factors
 rRNA precursors - own removal (self-splicing)
 mRNA precursors – spliceosome, composed of snRNP and many more
additional proteins

24. Posttranscriptional Modification of mRNA
RNA splicing
 mRNA precursors – spliceosome, composed of snRNP and many
more additional proteins
 snRNP removes intervening sequences from pre-mRNA

25. NONCODING RNAs (ncRNA)
Divided into:
 Long ncRNA – lncRNA
 Involved in epigenetic regulation of protein-coding gene expression
and modulation of gene transcription and protein degration
 Small ncRNA – microRNA (miRNA)
 Endogenous to the cell and produced by transcription of the cell’s
gene
 Small interfering RNA (siRNA)
 Exogenous sources such as a viral infection or synthetic dsRNA
 Small nucleolar RNAs (snoRNAs) & Small nuclear RNA (snRNAs)

26. NONCODING RNAs (ncRNA)
mRNA degradation mechanism directed by siRNA
 Dicer, one of the RNAse III endonucleases, takes dsRNAs and cut
them to their characteristic small size leaving a two nucleotide-long
3’ overhang
 One RNA is cleaved, it passes to a protein complex RNA-induced
silencing complex (RISC)
 In an ATP-dependent process, RISC unwinds the dsRNA and selects
the antisense strand
 Argonaut, a nuclease protein, guides the complex to the targeted
mRNA
 siRNA matching of the antisense strand to the mRNA is perfect and
can be cleaved then silenced

27. NONCODING RNAs (ncRNA)
mRNA degradation mechanism directed by miRNA
 Transcribe by RNA pol II from what are called MIR genes.
 First intermediate transcript are “hairpin” molecules called pri-miR
 Pre-miRs are transported to the cytoplasm by exportin-5 and the
cofactor Ran-GTP
 Once in the cytoplasm, pre-miRs are processed by the enzyme dicer.
Dicer binds to the pre-miRNA and cleaves it to miRNA
 miRNA binds to RISC as it did to with siRNA. They are guided to the
complementary sequence of the target mRNA

28. NONCODING RNAs (ncRNA)
mRNA degradation mechanism directed by miRNA
 If the process is complete, the mRNA will be degraded. In the
miRNA is not a perfect match for the mRNA target, there is no
cleavage of mRNA, but the RISC continues to bind to the mRNA,
interfering with the ability of the ribosomes to translate mRNA

29. NONCODING RNAs (ncRNA)
Both siRNA and miRNA can Repress transcription
 can repress through the association with the RNA-induced initiation
of transcription silencing complex (RITS)
 The antisense RNA strand within the RITS targets the RITS complex
to specific gene promoters or large regions of chromatin
 RITS then recruits chromatin remodeling enzymes to the promoters
and these enzymes methylate histones and DNA which will result to
heterochromatin formation and subsequent transcriptional
silencing

30. THANK YOU!!