Adobe Systems
Department of Experimental Biology
1
Bi9325
Molekulární genetika člověka
Mgr. Jiří Kohoutek, Ph.D.

Adobe Systems
2
Lecture 7
• Regulation of gene expression in prokaryotes and eukaryotes.
Department of Experimental Biology

Adobe Systems
3
Why to regulate gene expression?
•The products of all genome genes are not necessary at every moment of a cell's life - mechanisms
to ensure gene expression at the right time and in the right place (spatiotemporal regulation).
•
•Variability of the external environment. Responses to signals from the environment (e.g.
temperature, osmotic pressure, nutrient availability,..) and signals from other cells, tissues and
organs (e.g. developmental processes, injuries,..).
•
•Variability of the internal environment of the cell during the cell cycle, cell commitment,
differentiation and etc.
•
•Gene expression is highly energetic process.
Department of Experimental Biology

Adobe Systems
Define footer – presentation title / department
4
• Regulation of gene expression in the eukaryotes.

Adobe Systems
5
•Eukaryotic gen expression (like us!) can be controlled at various stages, from the availability of
DNA to the production of mRNAs to the translation and processing of proteins.
•
•Different genes are regulated at different points, and it’s not uncommon for a gene (particularly
an important or powerful one) to be regulated at multiple steps.
•
•Chromatin accessibility.
•Transcription.
•RNA processing.
•RNA stability.
•Translation.
•Protein activity.
Department of Experimental Biology
Eukaryotic gene expression
https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/regulation-of-gene-ex
pression-and-cell-specialization/a/overview-of-eukaryotic-gene-regulation

Adobe Systems
6
•Enhancer/Silencer – DNA control element far from or close a gene or intron.
•
•Activators – bind to enhancers to turn on transcription of a gene.
o Transcription factors.
o Needed for transcription to begin.
•
•Repressors – bind to silencers.
o Turn off transcription.
o Block activators from binding to enhancers.
Department of Experimental Biology
Components of eukaryotic gene expression
https://www.uwyo.edu › 13-miller-chap-7c-lecture

Adobe Systems
7
Department of Experimental Biology
Components of eukaryotic gene expression
•Co-activator and Co-repressor - the distinction between an activator/co-activator and
repressor/co-repressor is based on whether or not the protein binds specifically to DNA.
•
•Namely, activators/repressors have DNA binding domains that allow them to bind to DNA.
•
•Co-activators/co-repressors typically don’t bind to specific sequences in DNA.
•
•They typically exert their effects on transcription initiation via protein-protein interactions
within transcription initiation complexes at promoters, or by modifying histone tails.
https://www.uwyo.edu › 13-miller-chap-7c-lecture

Adobe Systems
8
Chromatin accessibility
•Heterochromatin is a condensed form that has a condensation state similar to chromatin found in
metaphase chromosomes. Heterochromatin typically is found at centromere and telomere regions, which
remain relatively condensed during interphase.
•
•Euchromatin is considerably less condensed. Most transcribed genes are located in regions of
euchromatin.
•Chromatin accessibility - the structure of chromatin (DNA and its organizing proteins) can be
regulated. More open or “relaxed” chromatin makes a gene more available for transcription.
•
•Interphase chromatin exists in two different condensation states.
Department of Experimental Biology
https://www.uwyo.edu › 13-miller-chap-7c-lecture

Adobe Systems
9
https://www.sciencedirect.com/topics/engineering/peptide-bond
Department of Experimental Biology
Chromatin accessibility
•Transition is between state of hetero- and euchromatin is mediated by various modifications of
histones.
Amino acids
available
for chemical
modification
Histone
tails
DNA
double
helix
Nucleosome
(end view)
(a) Histone tails protrude outward from a nucleosome
Unacetylated histones
Acetylated histones
(b)
Acetylation of histone tails promotes loose chromatin
structure that permits transcription

Adobe Systems
10
•The trimethylation of histone H3 at lysine 9 (H3K9Me3) plays an important role in promoting
chromatin condensation to heterochromatin.
•
•Trimethylated sites are bound by heterochromatin protein 1 (HP1) which self-associates and
oligomerizes resulting in heterochromatin. Heterochromatin condensation is thought to spread
laterally between “boundary elements” that mark the ends of transcriptionally active euchromatin.
•
•Recruitment of the H3K9 histone methyl transferase (HMT) to HP1 sites promotes heterochromatin
spreading by catalyzing H3 methylation.
•
Department of Experimental Biology
Euchromatin to heterochromatin transition
https://www.uwyo.edu › 13-miller-chap-7c-lecture

Adobe Systems
11
Department of Experimental Biology
Euchromatin to heterochromatin transition
•RPD3 is a histone deacetylase, an enzyme removing acetyl groups from histones in the vicinity of
the URS1 sequence. The nucleosomes bound to DNA in this region (which contains a TATA box promoter)
subsequently condense, and expression of the gene is repressed.
•Another mechanism involved in heterochromatin formation from euchromatin is direct histone
deacetylation.
•
•The UME6 repressor binds to URS1 control elements and recruits a co-repressor complex containing
SIN3 and RPD3 to these sites.
https://www.uwyo.edu › 13-miller-chap-7c-lecture

Adobe Systems
12
Department of Experimental Biology
•The GCN5 acetylates histone N-terminal tails.
•
•Hyperacetylation of histones leads to chromatin decondensation.
•
•General TFs and RNA Pol II are then able to interact with the promoter, and the gene is
transcribed.
Histone acetylation
•In general, the genes can be turned on by histone acetylation and chromatin decondensation.
•
•For instance, GCN4 activator first binds to its UAS upstream of the TATA box of a regulated gene
and recruits a co-activator complex containing the GCN5.
https://www.uwyo.edu › 13-miller-chap-7c-lecture

Adobe Systems
13
•Cytosine methylation in higher eukaryotes: 10-30%.
•
•Mediated by DNA methyltransferases (DNMTs).
•
•Targeted sequence is short: GC in animals and GNC in plants.
•
•DNA methylation typically weakens gene expression.
•
•Genes with continuous transcription mostly do not have GC methylated islands.
•
•Methyl groups protrude into a large DNA groove „ thus preventing proper binding of transcription
factors“.
Department of Experimental Biology
DNA Methylation

Adobe Systems
14
Department of Experimental Biology
•The process of DNA methylation involves the transfer of methyl group from S-adenosylmethionine
(SAM) to the C-5 position of cytosine, catalyzed by DNA methyltransferases.
•
•DNA methylation is an "epigenetic switch" that regulates the balance between "open" and "closed"
form of chromatin by changing the interactions between DNA and protein.
DNA Methylation
Pharmacology & Therapeutics (2018), https://doi.org/10.1016/j.pharmthera.2018.02.006

Adobe Systems
15
Transcription - activity of eukaryotic cell
•Transcription is a key regulatory point for many genes. Sets of transcription factor proteins bind
to specific DNA sequences in or near a gene and promote or repress its transcription into the RNA.
•
•Unlike prokaryotes, eukaryotic genes are not completely turned on or off, but there is modulation
of transcription.
•
•Basal transcription - with the participation of basal TF, transcriptional levels, minimum level of
transcription.
•
•Constitutive transcription - with the participation of basal and constitutive TFs, allowing
different transcription rates of different gene.
o General TF = basal + constitutive, activate operational genes
•
•Induced transcription - transcription regulated by inducible specific TFs which activity is
influenced by stimuli from the external or internal environment.
o Specific TF= cellular and time-specific regulatory proteins.
Department of Experimental Biology

Adobe Systems
16
Department of Experimental Biology
https://www.khanacademy.org/science/biology/macromolecules/proteins-and-amino-acids/a/orders-of-pro
tein-structure
Requirements for transcription initiation
•Putting RNA-polymerase in the active state.
•
•Binding of TF to the promoter (with the participation of activators and coactivators, necessary to
create a pre-initiation complex.
•
•Binding of specific (inducible) TFs to transcription enhancers with unique response sequences
(RE).
•
•TF interaction allowing the transcription promoter and enhancer to interact.
•
•Active RNA polymerase state.

Adobe Systems
17
•The promoter of a eukaryotic gene can be defined as a sequence that sets the transcription start
site for RNA polymerase.
•Strong RNA Pol II promoters contain an A/T rich sequence known as the TATA box located 26-31 bp
upstream of the start site.
•Other genes have alternative sequence elements known as initiators (Inr) which also serve as
promoters that set the RNA Pol II start site.
•Finally, CG-rich repeat sequences (CpG islands, BRE) are used by RNA Pol II as promoters in 60-70%
of genes. Most of these genes are weakly expressed.
•
Department of Experimental Biology
Promoter structure
https://view.officeapps.live.com/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTranscriptions
lide.ppt&wdOrigin=BROWSELINK

Adobe Systems
18
•Basal (general) transcription factors.
•
•Architectural regulators to facilitate DNA looping.
•
•Transcription factors, transcription activators.
o Proteins that bind to upstream or downstream activator sequences (UASs).
•Chromatin modification/remodeling proteins.
•
•Coactivators
o Act indirectly (with other proteins, not with DNA).
Department of Experimental Biology
RNA polymerase lI require five types of proteins

Adobe Systems
19
Transcription initiation by RNA Pol II
•RNA Polymerase II (RNA Pol II) requires general TFs in addition to tissue-specific transcription
factors for transcription of most genes in vivo.
•
•General transcription factors, TFs, position RNA Pol II at start sites and assist the enzyme in
melting promoter DNA. General TFs are highly conserved across species. The general TFs used at TATA
box.
•
•Architectural regulators facilitates DNA looping. Such as, TFIID consists of TBP (TATA box binding
protein) and 13 TBP-associated factors (TAFs).
Department of Experimental Biology
https://view.officeapps.live.com/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTranscriptions
lide.ppt&wdOrigin=BROWSELINK

Adobe Systems
20
•Provide a response to various extracellular or intracellular stimuli indicating the need to turn
on one or more genes.
•
•Unlike most proteins, they are able to enter the nucleus.
•
•Recognize and bind to specific DNA sequences.
•
•Make contact with the transcription apparatus, either directly or indirectly.
Department of Experimental Biology
Characteristics of transcription factors (TF)

Adobe Systems
21
•A given DNA-binding motif can occur in a number of proteins where it carries out the same or
similar functions.
•
•For examples of the coiled-coil, EF hand/helix-loop-helix, and zinc-finger motifs.
Department of Experimental Biology
Characteristics of transcription factors (TF)
https://view.officeapps.live.com/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTranscriptions
lide.ppt&wdOrigin=BROWSELINK
•DNA-binding domains of several transcription factors employ similar tertiary structure – motif.
•DNA- binding motifs are evolutionarily conserved AA sequence which have a defined conformation
since AA sequence ultimately determines structure.

Adobe Systems
22
•DNA-binding motifs bind specifically to DNA via non-covalent interactions.
•
•Helix – Turn – Helix. The second helix in this motif (the DNA recognition helix) typically binds
to a specific sequence of bases in DNA.
•
•Alpha-helices are one of the most common types of DNA-binding sequences. The side-chains of
residues within the α-helix often bind to the surfaces of bases exposed in the major groove of
double-helical DNA. Binding to phosphates and bases in the minor groove typically is less
important.
•Helix-turn-helix TFs are common in bacteria.
Department of Experimental Biology
DNA-binding motifs in TFs
https://view.officeapps.live.com/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTranscriptions
lide.ppt&wdOrigin=BROWSELINK

Adobe Systems
23
Department of Experimental Biology
•Most TFs containing this motif are dimeric. Nuclear receptors, which bind steroid hormones and
other compounds, contain this motif. Zinc ions are bound to the DNA recognition helix of this
motif, which contacts bases in the major groove.
DNA-binding motifs in TFs
•Zinc finger - most common DNA-binding motif in human and multicellular animal.
•
Two types of zinc finger TFs:
•C2H2 zinc finger TFs - its 2 cysteine and 2 histidine residues bind to zinc ions (Zn2+) and the
α-helix containing the 2 histidines binds to bases in the major groove.
•
•C4 zinc finger TFs is much less common.
A schematic illustrating the parts of zinc fingers. Starting from the left is burgundy coil with a
thread-like structure hanging and curled to its left; this is labeled as zinc finger motif, and
shows a small purple ball intertwined in the threads. The ball is labeled Zn [superscript 2+]. To
the right of the burgundy thread is a red coil, which also has thread-like loops to its top right;
this is labeled as zinc finger motif, and also has a purple zinc ball lodged into the loop. In
between these two sections are two large blue strands that form a curved X with blue hexagons
hanging on them. To the right, one of the blue strands connects to a pink thread-like structure
looped a few times on top of itself. It too has a purple zinc ball lodged into it, as well as a
pink arrow pointing up. To the left of the ball is a pink and purple hexagon, also hanging; this is
labeled as His. Below the ball is a pink thread that curves from the arrow to the right of the
ball, and has some burgundy sections in it.; this is labeled as Cys. The entire pink structure is
labeled as zinc finger motif.
https://www.coursehero.com/Lehninger_Ch28.ppt - 28, Regulation of Gene Expression

Adobe Systems
24
•Leucine-zipper TFs
•
•Contain extended a-helices wherein every 7th amino acid is leucine. This periodicity creates a
nonpolar face on one side of the helix that is ideal for dimerization with another such protein via
a coiled-coil motif.
•
•So-called basic zipper (bZip) TFs have a similar structure except that some leucines are replaced
by other nonpolar amino acids.
•
•The N-terminal ends of both leucine-zipper and bZip proteins contain basic amino acids that
interact with bases in the major groove.
Department of Experimental Biology
DNA-binding motifs in TFs
Basic residues
https://view.officeapps.live.com/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTranscriptions
lide.ppt&wdOrigin=BROWSELINK

Adobe Systems
25
•Helix – Loop – Helix TF
•
•Another class of TF, the basic helix-loop-helix (bHLH) proteins are similar to bZip proteins, but
contain a loop between the DNA recognition helix and the coiled-coil region. bZip and bHLH proteins
commonly form heterodimeric TFs.
Department of Experimental Biology
DNA-binding motifs in TFs
https://view.officeapps.live.com/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTranscriptions
lide.ppt&wdOrigin=BROWSELINK
Basic residues
A molecular structure illustrating specific amino acid residue-base pair interactions; the first
one shows Thymine double bonded to Adenine, and the second structure shows Cytosine triple bonded
to Guanine. A molecular structure illustrating specific amino acid residue-base pair interactions;
the first one shows thymine double-bonded to adenine, and the second structure shows cytosine
triple-bonded to guanine. The first structure is labeled as glutamine (or asparagine) in pink.
Starting from the bottom left is a six-membered ring with an N at the bottom left point. N bonds
down and left. Moving counterclockwise on the structure, the next point double bonds down and right
to O. The next point, which is the middle right point, has an N; the top right point double bonds
up and right to O, and the top left point bonds up and left to CH3. The N at the middle right point
bonds right and slightly down to H, which has a blue-dashed bond down and right to N on another
six-membered ring with alternating double bonds and with the top point labeled 6. The bottom point
on this ring also has an N. The top point, labeled 6, bonds up and left to N, which bonds up and
left H that has a blue-dashed bond to the O at the top right point of the first ring. The N also
bonds up and slightly right to H, which blue-dashed bonds up and right to (all pink starting from
this point) O, which double bonds up and right to C. C bonds up to CH2, which bonds up to CH2,
which bonds up to C, which bonds left to N, which bonds down to H and left to R. The C also bonds
up to H and right to C, which double bonds up to O and right to R’. Going back to the first C in
the pink portion of the structure, this C bonds down and right to N, which bonds down and right to
H and down and left to H (these are also in pink). The H blue-dash bonds down to an N, labeled 7,
as the top point on a five-membered ring. The side to the right of the N is double bonded, and the
bottom right point on the ring also has an N that bonds down and right. The five-membered ring
shares a double bond on its left side with the six-membered ring labeled 6 at the top (previously
described). To the right of all this, pink text is arginine (pink). To the right of this structure
is the cytosine-guanine triple bond structure. Starting at the bottom left is a six-membered ring
with an N at the bottom left point, which bonds down and left. The top left side of the ring is
double bonded, and the side to the right of the top point is also double-bonded down to an N on the
ring. The bottom right point of the ring double bonds down and right to O, and the top right point
bonds up and right to N, which bonds up and left to H, and slightly down and right to H. The O on
the structure blue-dash bonds right and down to H, which bonds right and slightly down to N, which
bonds down and right to H and up and right to a six-membered ring labeled 6 at the top left point.
The top left of the structure is N, which bonds up and left to H, which blue-dash bonds left to the
N at the top right of the first ring. The top part of the ring labeled 6 double bonds up and left
to an O, which blue-dash bonds up and left to H that bonds up and right to the N that bonds down to
the first ring. The bottom left side of the ring is double bonded, and the right side of the ring
is double bonded right to a five-membered ring, which has a double-bonded top right side and has N
at the top point and N at the bottom right point, which bonds down and right. This same O blue-dash
bonds up and right to (all in pink starting from this point) H, which bond up to N, which bonds
left to H and up and right to C, which bonds down and right to N, which bonds down to H and right
to H. The H bonding down blue-dashes down to N at the top pf the 5-membered ring labeled as 7. The
N-C-N bonds have a dashed pink line below them with a plus sign below the C. The C bonds up to NH,
which bonds up to CH2, which bonds up to CH2, which bonds up to CH2, which bonds up to C. C bonds
left to N, which bonds left to R and down to H. The same C bonds up to H and right to C, which
double bonds up to O and right to R’.
https://www.coursehero.com/Lehninger_Ch28.ppt - 28, Regulation of Gene Expression

Adobe Systems
26
 RNA Pol II preinitiation complex formation
•The sequential steps leading to the assembly of the RNA Pol II pre-initiation complex.
•
•1. TBP binds to the TATA box and bends (DNA looping) DNA near the promoter.
•
•2. TFIIB binds, and then a complex between Pol II and TFIIF loads onto the promoter.
•
•3. TFIIF positions the Pol II active site at the mRNA start site and helps maintain chromatin at
the promoter in an uncondensed state.
•
•4. TFIIE then binds creating a TFIIH docking site.
•
Department of Experimental Biology
https://view.officeapps.live.com/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTranscriptions
lide.ppt&wdOrigin=BROWSELINK

Adobe Systems
27
Department of Experimental Biology
https://view.officeapps.live.com/op/view.aspx?src=http%3A%2F%2Fwww.hixonparvo.info%2FTranscriptions
lide.ppt&wdOrigin=BROWSELINK
 RNA Pol II preinitiation complex formation
•5. With the addition of TFIIH, the assembly of the pre-initiation complex is complete.
•
•6. Subsequently, one subunit of TFIIH melts DNA at the promoter, obtaining energy by ATP
hydrolysis. RNA Pol II then begins transcribing the mRNA.
•
•Another subunit of TFIIH phosphorylates the RNA Pol II CTD, making RNA Pol II highly processive.
•
•Tissue-specific TFs bound to enhancers and promoter-proximal elements also play important roles in
transcription initiation in vivo.
•
•

Adobe Systems
28
Department of Experimental Biology
Transcription – elongation phase
Cell, 2011, Levine M., May 13, 145
Figure thumbnail gr3
•RNA Pol II undergone promoter escape and contains phosphorylation of serine 5 (Ser5) in the
C-terminal domain (CTD).
•
•Recruitment of P-TEFb (positive transcription elongation factor b) causes phosphorylation of Ser2
in the CTD, resulting in RNA Pol II transcription elongation.
•Right after transcription initiation the RNA polymerase II is paused typically 30–50 nucleotides
after transcription initiation site.
•Negative transcription elongation factors DSIF, NELF.

Adobe Systems
29
•Posttranslational modification, phosphorylation in particular, of CTD of RNA Pol II corresponds
with specific phase of transcription.
•
•Other modification of CTD of RNA Pol II.
o Acetylation
o Proline isomerization.
•
•We talk about CTD code.
•
Department of Experimental Biology
Transcription – elongation phase
 Trends Genet. 2012 Jul;28(7):333-41.

Adobe Systems
30
•Modification of CTD of RNA Pol II coincides with various steps in transcription.
•
o DNA and chromatin remodeling.
o
o Histone modifications.
o
o DNA processing.
o
o Transcription termination and polyadenylation.
Department of Experimental Biology
Transcription – elongation phase
 Trends Genet. 2012 Jul;28(7):333-41.

Adobe Systems
31
Enhancers and Activators
•Distal control DNA elements, which are called enhancers, may be far away from a gene or even
located in an intron.
•
•Enhancers can be thousands of nucleotides away from the TATA box of the promoter.
•
•Can be bound by activators.
•
•Activators have two domains DNA-binding, protein-binding, and/or signal molecule-binding domains
coupled with transcription activating domain.
•
•Facilitate a sequence of protein-protein interactions that result in transcription of a given
gene.
•
•Some regulate a few genes; some regulate many hundreds of genes.
Department of Experimental Biology
https://www.camphillsd.k12.pa.us/ short chap 18.ppt
https://ww2.chemistry.gatech.edu/Lehninger_Ch28.ppt

Adobe Systems
32
Department of Experimental Biology
Enhancers and Activators
https://www.camphillsd.k12.pa.us/ short chap 18.ppt
Activators
DNA
Enhancer
Distal control
element
Promoter
Gene
TATA box
General
transcription
factors
DNA-
bending
protein
Group of mediator proteins
RNA
polymerase II
RNA
polymerase II
RNA synthesis
Transcription
initiation complex

Adobe Systems
33
Department of Experimental Biology
Chromatin remodeling complexes
•CRE - cAMP response element.
•
•CREB - (cAMP response element-binding protein/CRE-binding protein).
o Specific transcription factor.
o Transcription of genes regulated by CREB: somatostatin, c-fos, tyrosine hydroxylase,
neuropeptides, enkephalin, genes involved in circadian rhythm control, and more.
•
•CBP–CREB binding protein.
o Histon acetyltransferase.
o Coactivator CREB.
o Many other transcription factors (c-myb, c-fos, p53, E2F, NF-κB,...).

Adobe Systems
34
European Respiratory Journal 2005 25: 552-563
Department of Experimental Biology
Chromatin remodeling complexes

Adobe Systems
Define footer – presentation title / department
35
European Respiratory Journal 2005 25: 552-563
Chromatin remodeling complexes

Adobe Systems
36
Department of Experimental Biology
•Mediator is an ∼30-subunit multiprotein complex.
•
•Mediator functions as a molecular bridge between RNA Pol II and transcription factors bound to
enhancers and promoter proximal transcription control sequences.
•Mediator influences all stages of transcription – from initiation to elongation.
•
•Non-coding RNA interacts directly with Mediator to influence transcription.
PNAS, 2012, 109 (48) 19519-19520
Mediator complex
https://en.wikipedia.org/wiki/Mediator_(coactivator)

Adobe Systems
37
•The mammalian Mediator complex interacts directly with a subset of long noncoding RNAs termed
lncRNA-activating (ncRNA-a), which activate neighboring genes using a cis-mediated mechanism.
Department of Experimental Biology
Mediator complex
Figure thumbnail gr1 Figure thumbnail gr3
Trends in Biochemical Sciences, Nov 2013, Vol. 3, Is. 11, P531-537
•It can activate or repress transcription.

Adobe Systems
38
Department of Experimental Biology
RNA processing
•Splicing, capping, and addition of a poly-A tail to an RNA molecule can be regulated.
•
•Different mRNAs may be made from the same pre-mRNA by alternative splicing.
https://courses.lumenlearning.com/wm-biology1/chapter/reading-post-translational-control-of-gene-ex
pression/
A pre-mRNA has four exons separated by three introns. The pre-mRNA can be alternatively spliced to
create two different proteins, each with three exons. One protein contains exons one, two, and
three. The other protein contains exons one, three and four.

Adobe Systems
39
•Alternative RNA splicing is a mechanism that allows different protein forms to be produced from
one gene when different combinations of introns, and sometimes exons, are removed from the
transcript.
•
•It is a common mechanism of gene regulation in eukaryotes; according to one estimate, 70 percent
of genes in humans are expressed as multiple proteins through alternative splicing.
•
•The cause of many genetic diseases is improper alternative splicing rather than mutations in a
sequence.
Department of Experimental Biology
Alternative splicing
https://courses.lumenlearning.com/wm-biology1/chapter/reading-post-translational-control-of-gene-ex
pression/
Diagram shows five methods of alternative splicing of pre-mRNA. When exon skipping occurs, an exon
is spliced out in one mature mRNA product and retained in another. When mutually exclusive exons
are present in the pre-mRNA, only one is retained in the mature mRNA. When an alternative 5′ donor
site is present, the location of the 5′ splice site is variable. When an alternative 3′ acceptor
site is present, the location of the 3′ splice site is variable. Intron retention results in an
intron being retained in one mature mRNA and spliced out in another.

Adobe Systems
40
Splicing Produces Related but Distinct Protein Isoforms
https://slideplayer.com/slide/5746723/
Department of Experimental Biology

Adobe Systems
41
RNA stability
Department of Experimental Biology
https://open.lib.umn.edu/evolutionbiology/chapter/5-8-using-the-genetic-code-2/
•RNA stability. The lifetime of an mRNA molecule in the cytosol affects how many proteins can be
made from it.
•
•Capping.
•
•Degradation of mRNA.
•
•Small regulatory RNAs called miRNAs can bind to target mRNAs and cause them to be chopped up.
•
•Small  nuclear RNA – 7SK RNA, U1snRNA.
•
•Long noncoding RNA.
•

Adobe Systems
42
Regulation of 5´- CAP
The 5′ cap has four main functions:
•Regulation of nuclear export of RNA is regulated by the cap binding complex (CBC), which binds to
7-methylguanylate-capped RNA. The CBC is then recognized by the nuclear pore complex and the mRNA
exported.
•Prevention of degradation by exonucleases.
•Promotion of translation.
•Promotion of 5′ proximal intron excision.
Department of Experimental Biology
https://wou.edu/chemistry/courses/online-chemistry-textbooks/ch450-and-ch451-biochemistry-defining-
life-at-the-molecular-level/chapter-10-transcription-and-rna-processing/

Adobe Systems
43
•In P-bodies the m7Gppp cap is removed by Dcp1/2 – dipeptydil carboxypeptidase 1 and 2.
•
•RNA without cap is degraded in the 5΄-to-3΄ direction by the Xrn1- 5´-3´exoribonuclease 1.
Department of Experimental Biology
Degradation of RNA
FEMS Yeast Research, Volume 18, Issue 6, September 2018, foy050,
Principal pathways of mRNA degradation in yeast. The first step of untranslated mRNA degradation is
deadenylation, operated by the CCR4/NOT complex also comprising Pop2, Caf4 and Caf130 proteins.
After that, deadenylated mRNAs are degraded in the 3΄-to-5΄ direction by the exosome complex or,
mainly, are transferred to P-bodies for decapping. In this process, mRNAs are bound by the Lsm1–7
complex, the Dhh1 helicase, the Edc1–3 and Dcp1/2 proteins and, after removal of the m7Gppp cap,
are degraded in the 5΄-to-3΄ direction by the exoribonuclease Xrn1. Pat1: deadenylation-dependent
mRNA-decapping factor, associated with topoisomerase II, also involved in regulation of
translational initiation; EIF4G, EIF4E: translation initiation factors; Pab1: poly(A)-binding
protein.
•The first step of untranslated mRNA degradation is deadenylation, removal of polyA tail.
•
•Deadenylated mRNAs are degraded in the 3΄-to-5΄ direction by the exosome complex or, mainly, are
transferred to P-bodies for decapping.

Adobe Systems
44
Department of Experimental Biology
miRNA
•18-25 nt, single-strand molecules.
•
•Post-transcriptionally regulate gene expression.
•
•2694 human miRNAs (miRBASE in 22, March 2018).
•
•Evolutionarily conserved and transcribed by RNA Pol II.
•
•1-2 % of the genome.
•
•Genes for miRNAs on all human chromosomes except Y.
•
•Regulation of the expression of up to 50% of protein-coding genes.
•
•One miRNA can regulate tens to hundreds of target mRNAs.
MicroRNA

Adobe Systems
45
Department of Experimental Biology
•A miRNA is first transcribed as a long RNA molecule, which forms base pairs with itself and folds
over to make a hairpin.
•
•Next, the hairpin is chopped up by Drosha enzymes, releasing a small double-stranded fragment of
about 22 nucleotides and processed by Dicer.
•
•One of the strands in this fragment is the mature miRNA, which binds to a specific protein to make
an RNA-protein complex RISC – RNA-induced silencing complex.
•
•Targeting of mRNA
o mRNA cleavage.
o Translation repression.
Biogenesis of miRNA

Adobe Systems
46
Department of Experimental Biology
•Physiological function
o Proliferation.
o Differentiation.
o Apoptosis and etc.
•
•Tumorigenesis.
https://cen.acs.org/articles/89/web/2011/04/Pyrrolysine-Synthesis-Revealed.html
Function of miRNA

Adobe Systems
47
•Postinitiation mechanisms (A).
•
•Cotranslational protein degradation (B).
•
•Initiation mechanisms. MicroRNAs interfere with a very early step of translation, prior to
elongation.
oArgonaute proteins compete with eIF4E for binding to the cap structure (C).
oArgonaute proteins recruit eIF6, which prevents the large ribosomal subunit from joining the small
subunit (D).
oArgonaute proteins prevent the formation of the closed loop mRNA configuration by an ill-defined
mechanism that includes deadenylation (E).
•
•MicroRNA-mediated mRNA decay. MicroRNAs trigger deadenylation and subsequent decapping of the mRNA
(F).
Department of Experimental Biology
Mechanism of miRNA-mediated gene silencing
Cell, Jan 2008, Vol. 132, Is. 1, P9-14.
Figure thumbnail gr1

Adobe Systems
Define footer – presentation title / department
48
•Small nuclear RNA (snRNA) is one of the small RNA with an average size of 150 nt.
•
•Eukaryotic genomes code for a variety of non-coding RNAs.
•
• snRNA is a class of highly abundant RNA, localized in the nucleus with important functions in
intron splicing and other RNA processing.
Small nuclear RNA
Figure 1
Trends in Genetics, Mar 2021, Vol. 37, Is. 3., P279-291.

Adobe Systems
49
Department of Experimental Biology
Long noncoding RNA – lncRNA
•Longer than 200 nt; encoded by genes on different chromosomes (in non-coding regions, even in gene
introns).
•
•Usually transcribed RNA polII (III), often have a cap at the 5'-end and a poly(A) at the 3'-end,
subject to splicing; do not have ORF, cannot be translated.
•
•Often tissue-specific and developmental stages specific.
•Some lncRNA found in specific DNA locus.
•Changes in the expression of lncRNAs associated with various diseases (cancer, Alzheimer's,
atherosclerosis), can also serve as markers.
•
•Present in body fluids, possibility of non-invasive diagnostics.
•
•In humans about 50,000 -100,000 genes for lncRNA, up to 270,000 different transcripts lncRNAs.

Adobe Systems
50
Department of Experimental Biology
Effect of lncRNA on gene expression
J Cell Mol Med. 2017 Dec;21(12):3120-3140.

Adobe Systems
51
Department of Experimental Biology
Effect of lncRNA on gene expression
•LncRNA Xist regulation network of genetic interactions.
Front. Cell Dev. Biol., 10 June 2021
•lncRNAs Xist regulates process of X-chromosome inactivation (XCI).
•
•lncRNA Xist recruits protein complexes to initiate, establish, and maintain the XCI state by
histone modifications, DNA methylation, and H4 hypoacetylation.

Adobe Systems
52
•Myc transcription.
Department of Experimental Biology
Role of lncRNA in tumorigenesis
Nature Reviews Molecular Cell Biology volume 22, pages96–118 (2021)
•Guardin – induced by p53 during DNA damage.
•
•Cytoplasm - sponge – stabilizes TRF2 mRNA
•Nucleus – enables interaction of BRCA1 and BARD to help recruit DSB machinery.

Adobe Systems
53
Department of Experimental Biology
•Translation of mRNA involves many “helper” proteins, which make sure the ribosome is correctly
positioned.
•
•Elongation initiation factor-2 (eIF-2) binds to a part of the ribosome called the small subunit.
•
•When eIF-2 is phosphorylated, it's turned "off„ - it undergoes a shape change and can no longer
play its role in initiation, so translation cannot begin. When eIF-2 is not phosphorylated, in
contrast, it's "on" and can carry out its role in initiation, allowing translation to proceed.
•
•In this way, phosphorylation of eIF-2 acts as a switch, turning translation on or off.
Regulation of translation

Adobe Systems
54
Signaling pathways in regulation of translation
•The mTOR and MAPK pathways affect the translatome by modulating the expression of specific subsets
of mRNAs.
•
•Phosphorylation of the 4E-BPs by mTOR leads to their dissociation from eIF4E, which stimulates the
interaction of eIF4E with eIF4G and assembly of the eIF4F complex.
•
•Phosphorylation of eIF4E also seems to bolster the translation of mRNAs encoding proteins involved
in tumor dissemination.
•
•Also eIF4A promotes the translation of mRNAs with G/C-rich 5′ UTR sequences, such as the
12-nucleotide guanine quartet (CGG)4 motif, which can form RNA G-quadruplex structures.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974435/
Department of Experimental Biology

Adobe Systems
55
https://www.cell.com/fulltext/S0960-9822(02)01135-1
Department of Experimental Biology
•The Ras/ERK and p38MAPK pathways are activated by a wide range of stimuli, including cytokines,
growth factors, and diverse environmental stresses.
•
•MNK interacts with eIF4G and phosphorylates eIF4E on Ser209, a site that increases its oncogenic
potential and facilitates the translation of specific mRNAs.
•
•RSK phosphorylates rpS6, eIF4B, PDCD4, and eEF2K, which are important regulators of translation.
•
•ERK and RSK also collaborate in the regulation of ribosome biogenesis by promoting TIF-1A
phosphorylation.
Signaling pathways in regulation of translation

Adobe Systems
56
https://steemit.com/science/@pjheinz/control-of-gene-expression-part-ii
Department of Experimental Biology
THANK YOU FOR YOUR  ATTENTION.