6.

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Gene Expression II: Transcription LE 6

Maria Milagros U. Magat, MD, MEM,FPPS, FPAPP 04/18/2018

OUTLINE
I. Overview of transcription
A. Definition of terms
B. Structure of RNA polymerase
C. Types of RNA polymerase
D. Structure of Prokaryotic and Eukatyotic mRNA
E. Compare and contrast Transcription and
Replication
II. Initiation (mRNA)
A. mRNA Promoter Regions
B. Initiation Complex Assembly
C. General Transcription Factors (GTF)
D. Gene Specific Transcription factors (GSTF)
E. Steps of initiation
F. Role of CTD in Transcription
G. Clinical Correlation: Xeroderma Pigmentosum
H. DNA Binding Domain and Mediator Proteins
III. Elongation mRNA
IV. Transcriptional modification
A. Co-transcriptional Modification
B. Post-transcriptional Modification
V. rRNA synthesis
A. Initiation
B. Post transcriptional Modification
C. Formation of Base modification Figure 1. Flowchart illustrating how genetic information encoded in DNA
VI. tRNA Synthesis produces proteins
A. Initiation
B. Posttranscriptional Modification  DNA codes have to be transcribed (copied) into RNA (mRNA,
C. Formation of Structure rRNA, & tRNA) first before they can be translated (produced)
D. Base Modification into proteins as the final gene products.
VII. Export to Cytoplasm
VIII. Decay of Normal Eukaryotic RNA  Transcription
A. Co-transcriptional Modification  The synthesis of RNA whose sequence is complementary to
B. Post-transcriptional Modification that of a DNAs template. Transcription begins at a promoter
IX. Regulation sequence and proceeds in the 5’ to 3’ direction. (Devlin)
A. Epigenetics: Chromatin Remodeling  Transcription is a complex process dependent on a major
B. Regulation of HMG CoA Reductase polymerase enzyme and a cast of supporting proteins.
X. Clinical Correlation: Metformin  Involves the Watson-Crick base pairing between
nucleotides of the template strand
LEARNING OBJECTIVES  Initial process of RNA synthesis
 Unique to transcription: There is co-transcriptional and
At the end of the lecture, the student should be able to: post-transcriptional modifications/processing of the
 To Compare and contrast: replication & transcription; primary transcript
prokaryotic & eukaryotic transcription; types of RNAP o Co-transcriptional modification – process of
 To Summarize: steps in eukaryotic RNA synthesis, co- and covalently altering the RNA sequence after the
post - transcriptional modifications, export to cytoplasm, and transcription begun but before the RNA has
decay been released from the RNA polymerase; while
 Conclude how regulatory (including epigenetic) mechanisms at transcription is occurring, the primary transcript
the transcriptional level affect gene expression of: is already being modified.
a. LDL-R o Post-transcriptional modification –
b. glucokinase modifications in the RNA after the transcription
c. PEPCK has been completed.
d. apoB48 & apo100  The DNA contains all the segments for the transcription of
 Summarize the role of metformin in the regulation of key different proteins required by the cell and the RNA
processes in metabolism polymerase utilizes these parts of the gene in order for
transcription to commence.
I. OVERVIEW OF TRANSCRIPTION
 Methylation
A. DEFINITION OF TERMS  Adding of methyl groups to the DNA or RNA molecule
 Central Dogma  Different effects in methylation of DNA as compared to
 Basic framework on how genetic information flows from a methylation of RNA
DNA sequence that stores information and determines the  In RNA: Vital component in production of biologically active
sequence of RNA, which in turn determines the structure of t/r/mRNA
the protein. (Devlin)  In DNA: Typically acts to repress gene expression

Trans Group 29 : Barbers, Belo, Buhat, Chan, de Guzman EDITOR: Andrea Cañete 1 of
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B. RNA POLYMERASE  Unlike DNA polymerase, no primer is required to initiate

 Enzyme that initiates transcription by binding to the promoter (a synthesis.
recognition sequence positioned in the gene), which signals the  Composed of:
site where RNA synthesis should commence.  2 α subunits which binds to the promoter DNA
 Opens up the DNA strand, which will result to a  1 β subunit which binds with the incoming nucleotides
transcription bubble needed for the process of  1 β’ subunit which aids in template binding
transcription (Clark)  1 σ subunit responsible specific transcription and the
o Critical in transcription: Accessibility of the selectivity of the gene to be transcribed
transcriptional complex to the promoter o Initially not present, but with binding of the core
region and the specific DNA region. Thus, it will become a holoenzyme.
opening up of the double-stranded DNA is o Essential for the correct initiation process
necessary. o Plays a regulatory function in the initiation of
 RNA Polymerase has catalytic properties and has Mg2+ in at the RNA transcription
center. This explains our requirement for magnesium especially
in repair, healing, growth, and development
 Catalyzes the formation of ester bonds between nucleotides
that base-pair with the complementary nucleotides in the DNA
template (Marks)
 Synthesis is always from a 5’ to 3’ direction, this would indicate
that RNA polymerase creates RNA based on the 3’ to 5’ strand
(anti-sense strand, non-coding strand or template strand)
 The sequence of the RNA is complementary to the antisense
strand (aka non-coding strand) of the DNA from which it is
synthesized. This means that the sequence of the new RNA
molecule is identical to the sense strand (aka non-template
strand) of the DNA. (Clark) – See figure below

Figure 4. RNA Polymerase σ subunit function in initiation process

C. TYPES OF RNA POLYMERASE

 RNA polymerase I (RNAP I for short)
Figure 2. RNA synthesis occurring from 5’ to 3’ direction based on the DNA
template strand
 Escherichia coli RNA Polymerase Structure
 Discovered by Roger D. Kornberg
 Transcribes rRNA (ribosomal RNA)
 Synthesized in the nucleolus
 rRNA: RNA component of ribosomes (protein required for
synthesis of other proteins; translation)
 Both eukaryotic and prokaryotic rRNA are composed of two
subunits based on the sedimentation coefficients
 Prokaryotic rRNA is composed of 50s & 30s subunits which
together form the 70s prokaryotic rRNA
 Eukaryotic rRNA is composed of 60s and 40s subunits
forming the 80s eukaryotic RNA

Figure 3. E coli RNA polymerase Structure

 RNA Polymerase Core Structure

 Capable of faithful transcription but not specific to RNA
synthesis Figure 5. Prokaryotic vs Eukaryotic rRNA differing in sedimentation
 Has both general and specific transcription factors that coefficients.
regulate it
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 RNA polymerase II (RNAP II)
 Transcribes mRNA (messenger RNA) and miRNA (micro  Most of the general aspects of the mechanism of the
RNA) in the nucleus transcription process in prokaryotes and eukaryotes are the
 Has both general and specific transcription factors that same, but there are several notable differences:
regulate it 1. Transcription in eukaryotes occurs within the nucleus under
 mRNA: moves genetic information from the DNA to the the direction of three separate forms of RNA polymerase
ribosome for translation; conveys genetic information from (RNAP I,II,III). Unlike the prokaryotic process, in
DNA to ribosome where specification of Amino acid eukaryotes the RNA transcript is not free to associate with
sequence occurs ribosomes prior to the completion of transcription. For the
 miRNA: Form of post-transcriptional gene regulation, mRNA to be translated, it must move out of the nucleus
specifically gene silencing/ down regulation into the cytoplasm
2. Initiation of transcription of eukaryotic genes requires the
 RNA polymerase III (RNAP III) compact chromatin fiber, characterized by nucleosome
coiling, to be uncoiled and the DNA to be made accessible
 Transcribes tRNA (transfer RNA) and other small RNAs
to RNA polymerase and other regulatory proteins. This
(snRNA and 5s RNA)
transition, referred to as chromatin remodeling, reflects the
dynamics involved in the conformational change that
Note: Prokaryotes only have RNA Polymerase I (RNAP I) and occurs as the DNA helix is opened.
serve all the functions of the three RNAPs. On the other hand, 3. Initiation and regulation of transcription in eukaryotes are
eukaryotes display all three forms of RNAPs. more complex as compared to prokaryotes. Eukaryotic
RNA polymerases, for example, rely on transcription
factors (TFs) to scan and bind to DNA. In addition to
Table 1. RNA polymerase to corresponding products promoters, other control units called enhancers and
RNA POLYMERASE Product repressors, may be located in the 5’ regulatory region
RNA Polymerase I rRNA upstream from the initiation point, but they have also been
RNA Polymerase II mRNA + microRNA found within the gene or even in the 3’ downstream region,
RNA Polymerase III tRNA +other small RNA (snRNA beyond the coding sequence.
and 5s RNA) 4. Alteration of the primary RNA transcript to produce mature
eukaryotic mRNA involves many complex stages referred
to generally as “processing.” An initial processing step
D. STRUCTURE OF PROKARYOTIC AND EUKARYOTIC involves the addition of a 5’ cap and a 3’ tail to most
transcripts destined to become mature mRNAs.
mRNA

E. COMPARE AND CONTRAST REPLICATION AND

TRANSCRIPTION
Table 2. Difference between replication and transcription
Replication Transcription
Template DNA parent DNA Region
strand
Product 2 stranded DNA 1 single stranded
RNA
Enzymes DNA polymerase RNA polymerase
Basal Transcription Not present Present
factors
Primer use Required Not needed
Site of Reactions Product remains Nucleus then
in nucleus translocated to the
cytoplasm
Directionality Bidirectional Unidirectional

Figure 6. Protoype Structure of prokaryotic and eukaryotic mRNAs;

Polycistronic mRNA vs Monocistronic mRNA
 Prokaryotic mRNA – polycistronic
 Polycistronic means that one mRNA strand can code several
different protein molecules.
 Have several sites for initiation and termination of codons
 Eukaryotic mRNA – monocistrionic
 Monocistronic means that one mRNA strand contains the
genetic information to translate only a single protein
 There is only one site for initiation and termination of protein
synthesis.
 Note: YGF in the figure means: Your Favorite Gene (Magat)
BIOCHEMISTRY
II. INITIATION
 Upstream region – region before the point of reference
(reference point: start point of mRNA transcription);
Downstream region – after the reference point in the linear
DNA
 In downstream everything will be transcribed including the
initiator region – Doc Magat
 Transcription begins at the end of the promoter, designated +1,
and continues until the end of the gene.
 At the upstream region (5’-end) is the promoter (to which the
RNA polymerase will attach)
 Downstream region begins with the information of the 5’-UTR,
the gene, then 3’-UTR (UTR means untranslated. Thus, these
are the regions in the mRNA that are not translated into
proteins)
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 5’-UTR and 3’-UTR regions are not used to make the final
protein, but often contain important regulatory elements
Figure 7. Upstream and Downstream Regions. The mature mRNA has
a methylated 5’ cap, a 5’-UTR, a protein coding sequence and 3’-
UTR and a polyA tail – Doc Magat
 Eukaryotic initiation
 Recognizing the promoter region of the DNA are various
transcription factors. Transcription factor facilitate the binding
of RNA Polymerase and therefore facilitates the initiation of
transcription. There are two categories of transcription
factors:
 General transcription factors (GTFs)- needed for
the transcription of all genes transcribed by a
specific RNAP; they are absolutely required for all
RNAP–mediated transcription
 Gene specific transcription factors (GSTFs) –
acts like “enzymes” since they speed up the
process of transcription. If there are no GSTFs,
transcription will proceed at a basal rate or very
slow rate and this means that the process of gene
expression may not be able to respond with the
need of the cell – Doc Magat
 Enhancer Regions
 Located far away from the gene
 When bound by GSTFs (specifically activators), the speed of
transcription is increased (explains the term “enhancer”)
 There are enhancers with mediator complexes attached to
them far away from the promoter which come in direct with a
mediator complex in the initiation complex
 DNA loops, allowing enhancers to come into contact with
bound transcription factors
Figure 8. Illustration of enhancer and DNA loops
BIOCHEMISTRY
A. PROMOTER REGION
 This is the region where the GTFs would assemble before RNA
polymerase II (RNAP II) would attach itself in a clocklike
manner – Doc Magat
 Contain recognition sequences
 Found upstream (in the upstream: there are some GTFs and
DNA Binding Domain (DBD) of the Gene Specific Transcription
Factor (GSTF) – Doc Magat
 Promoter Region is the reference site of upstream and
everything else is downstream –Doc Magat
 Upstream has participation, but not to be transcribed – Doc
Magat
 Where RNA polymerase attaches
 Because the interaction of promoters with RNA polymerase
governs the efficiency of transcription—by regulating the
initiation of transcription—the importance of promoter
sequences cannot be overemphasized.
 Consensus sequences represents the predominant bases found
within the first base transcribed in the mRNA and its respective
recognition sequences
 Prokaryotes
 The promoter region recognized by the sigma subunit of
bacterial RNA polymerase contains two special recognition
sequences approximately -10 and -35 nucleotides upstream
from the transcription start site
 The recognition sequence at -10 has the consensus
sequence TATAAT also called the Pribnow Box
 The recognition sequence at -35 has the consensus
sequence TTGACA
 Eukaryotes
 Sequence of three promoter regions that exist for RNA
polymerases to recognize:
 Upstream elements
 TATA box: binding site for a transcription factor that
guides RNA polymerase II to the promoter in eukaryotes
and is needed for binding of RNA polymerase (counter
part of pribnow box in prokaroytes)
 Initiator box: where transcription starts. Its consensus
sequence is YYCAYYYYY (-25 upstream from the TATA
box)
 Usually the initiator region is already downstream so
that it will be transcribed – Doc Magat
Figure 9. Eukaryotic Promoter Components – Initiator, upstream elements,
and TATA boxes. The promoter for RNA polymerase II has an initiator box
at the start site and a TATA box slightly upstream of this. Further upstream
are normally several upstream elements. The initiator box is a sequence
found at the site where transcription stars, and the TATA box is critical
sequence that allows RNA polymerase II to recognize the promoter (Clark)
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Figure 10. Idealized version of a promoter comprising various elements.

General consensus for the promoter region reference site is the TATA rich
region. GC box is a site for repressing gene expression by methylation.

B. INITIATION COMPLEX ASSEMBLY Figure 11. The initiation complex

C. GENERAL TRANSCRIPTION FACTORS (GTFs)

 Proteins that are required for transcription of all genes, as
opposed to specific transcription factors which are specific to
the gene being transcribed
 Can initiate transcription, however without specific transcription
factors, transcription proceeds at a very slow pace and
becomes incompatible with life.

Table 3. Eukaryotic General Transcription Factors

Transcription Factor Function
TBP (TATA Binding Protein) Component of TFIID that
recognizes the TATA box;
FIRST that binds to the TATA
box (REMEMBER!)
TFIID Binds to TATA Box;
Recruits TFIIB then TFIIA;
Largest
TFIIB Binds downstream the TATA
box and guides RNA Pol II in
place

TFIIA Binds upstream the TATA

box and guides RNA Pol II in
Figure 6. mRNA synthesis initiation complex (Holoenzyme vs Stepwise)
place;
 2 concepts (Devlin) dependent on the cell’s needs: Stabilizes TFIID and TFIIB
 Holoenzyme Model – Assembly as a holoenzyme of the TFIIF Accompanies and facilitates
general transcription factors and RNAP II before attaching to binding of RNA Pol II;
the gene and the promoter region Helps RNAP target its
 Stepwise Model – The stepwise assembly of the enzyme promoters
after the first or the initial components of the initiation factors
have attached to the promoter region TFIIE Helps recruit TFIIH;
Required for promoter
 The initiation complex is a set of assembled proteins necessary clearance and elongation
for transcription in eukaryotes (in prokaryotes the σ factor is the TFIIJ Required for promoter
only transcription factor required for transcription) clearance and elongation
 General transcription factors and RNA polymerase II form the
initiation complex
TFIIH Phosphorylates the Carboxyl-
terminal domain (CTD);
Has DNA Helicase activity
(forms the transcription
bubble)  starts elongation

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C. GENE SPECIFIC TRANSCRIPTION FACTORS (GSTFs)

 It has a domain, the DNA Binding Domain (DBD), which is in
direct contact with the upstream control element of the gene.
The other end, the activator domain, is directly associatied to
the assembled GTFs, which enhances the transcription – Doc
Magat (See figure 11 for illustration)
 Not required for transcription, however without these, speed of
transcription is incompatible with life
 May be activators or repressors
 Activators (See figure 8)
 Upregulate transcription
 May bind to enhancer regions
 Enhancer regions are DNA sequences upstream or
downstream the promoter region
 When an activator protein binds to an enhancer region a
conformational change occurs and the DNA bends
bringing the activator protein in contact to the mediator
protein
 Activator proteins may also interact with general
transcription factors to quicken the assembly of initiation
complex
 Repressors
 Functions similarly to activator proteins, but instead Figure 12. Binding of RNA polymerase II to promoter. Starting with TFIID,
downregulates transcription which contains TATA-binding protein (TBP), the components of the TFII
complex bind one after another to bind to DNA

D. STEPS IN INITIATION
1. TFIID binds to the TATA box promoter site via the TBP; binding
of TFIID distorts the sequence at the promoter region and this
serves as a landmark for other proteins
 TBP is found in three different protein complexes,
depending on whether RNA Pol I,II, or III is involved
 In the present case, TBP forms part of transcription factor
complex known as TFIID that is needed to recognize
promoters specific for RNA polymerase II
2. TFIIB is recruited to the promoter site by TFIID
3. TFIID then recruits TFIIA that stabilizes TFIID and TFIIB to the
promoter
 Here, the Preinitiation Complex (PIC) is formed.
4. The PIC will recruit TFIIF and RNA Pol II with a clamp
downstream. TFIIF helps RNAP II target its promoters
5. Once RNA Pol II is in place the complex is in a state called RNA
Pausing
6. Finally, TFIIE binds and recruits TFIIH forming the Closed
Complex
7. TFIIH and TFIIJ are recruited together. Upon recruitment, the
protein complex is called the closed complex
8. TFIIH has a DNA helicase activity, which splits the DNA strands
from each other, giving RNA Pol II access to the anti-sense strand,
at this stage the complex I called an Open Complex
 Recall the helicases separate the double-stranded DNA
into single strands allowing for the DNA template strand
to be transcribed Figure 13. RNA Pol II moves forward from the promoter.
9. TFIIH phosphorylates Carboxyl Terminal Domain (CTD)  end Before RNA pol II can move forward, the binding of other factors must
of initiation and start of elongation occur. One of these, TFIIH, phosphorylates the tail of RNA polymerase II.
The tail changes position with respect to the body of RNA polymerase II.
The other factors leave and RNA polymerase moves along the DNA and
begins the process of transcription

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E. ROLE OF CARBOXYL TERMINAL DOMAIN (CTD) IN
TRANSCRIPTION
 CTD – repetitive region at the C-terminus of RNA Pol II that may
be phosphorylated
 Contains the following amino acid sequence repeated 50x:
 YSPTSPS (tyrosine-serine-proline-threonine-serine-proline-
serine)
 Phosphorylated not only during initiation, but also during the
whole processes of transcription
Figure 14. Role of CTD in transcription
Phosphorylation of CTD
 CTD is phosphorylated by TFIIH most commonly at 5th serine
residue
 Phosphorylation of CTD releases RNA Pol II from the mediator
complex, allowing elongation to start
 The phosphorylated CTD recruits:
 Capping enzymes to prevent degradation of the mRNA at
the 5’-end
 Methyl transferases, which assists in the reformation of the
histone complexes which are removed during unwinding of
the DNA by helicase activity
 This recruitment allows from co-transcriptional modification of
the mRNA
 Marks the termination of the initiation and start of
Elongation, all the other GTFs have already dissociated
F. CLINICAL CORRELATION
 Xeroderma Pigmentosum
 Due to the defective phosphorylation of the CTD because of
the mutation of TFIIH  role of TFIIH is to phosphorylate
the CTD on the repeating amino acids
 Characterized by severe photosensitivity. Brief exposure to
sunlight would develop blisters, vesicular lesions on the skin
with burning sensation including appendages
 Very prone to dehydration, very painful and high incidence of
infection
G. DNA BINDING DOMAIN AND MEDIATOR PROTEIN
 DNA Binding Domain (DBD)
 Preset in Gene Specific Transcription Factors (GSTFs)
 Sites in the GSTFs that allow them to bind into specific DNA
sequences
 May be active or inactive depending on the conformation of
the transcription factors:
 Active: DBD is available
BIOCHEMISTRY
 Inactive: DBD is unavailable due to conformational
change
 A ligand may activate or inactivate the transcription factor,
similarly the loss of a ligand may activate or inactivate a
protein
 Mediator Protein
 Scaffold for RNA Pol II
 Primary function: integrate all regulatory signals by activator
or repressor proteins
 Upon activation, the RNA Pol is released from the mediator
protein and starts the process of elongation
III. ELONGATION
Figure 15. Elongation Process
 When RNAP is phosphorylated in the position of the promoter,
elongation takes place.
 Will commence only if the Carboxyl Terminal Domain (CTD)
is phosphorylated.
 Facilitated by TFIIH
 During elongation, a primary transcript is being formed in the
direction of 5’ to 3’
 During elongation, only TFIID remains with RNAP II while the
rest of the TFs dissociates. (Other sources would indicate the
TFIIH also remains with RNAP II
 Before RNA polymerase can move forward the binding of other
factors must occur. TFIIH phosphorylates the tail of RNAP II.
The tail changes position with respect to the body of RNA
polymerase II. The other factors leave and RNA polymerase
moves along the DNA and begins the process of transcription.
 While elongation is taking place, modifications in the RNA
molecule is also taking place due to the co-transcriptional
modifications that are present in the process of transcription.
 Methylation of internal nucleotides can occur. Note that the
methylation of RNA segments is normal in producing a
normal, biologically active RNA molecule.
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Transcription Bubble
Figure 16. Transcription Bubble
 Just like in replication (replication bubble), a transcription bubble
is present but only a portion of the DNA is transcribed.
 Inside the transcription bubble, there is a DNA-RNA hybrid.
 Elongation in transcription will only temporarily unwind the
strand & it will reform its helical structure (Magat)
Process of Elongation
 During the process, a primary transcript is being formed.
 The first 5’-ribonucleoside triphosphate, which is
complementary to the first nucleotide in the DNA template, is
inserted at the start site. As we noted, no primer is required.
Subsequent ribonucleotide complements are inserted and
linked together by phosphodiester bonds as RNA elongation
proceeds.
 The DNA being read is in the 3’-5’ direction, while the RNA
transcript being elongated is in the 5’-3’ direction
 A DNA-RNA hybrid is formed. DNA will become temporarily
be single-stranded while in the process.
 The phosphorylated CTD would enhance the arrival of 3
enzyme complexes: For CAPPING, POLYADENYLATION, &
SPLICING – these are for the modifications necessary to
make a mature RNA
Regulation
 In gene expression, regulation is typically the first step, however
in eukaryotic transcription, regulation occurs through the
attachment of Negative Elongation Factor (NELF) and DRB-
sensitivity inducing factor (DSIF).
 Negative regulation
 Once NELF & DSIF molecules attach to the transcription
complex, transcription stops.
 A cell in stress would have these two molecules hence,
stopping the transcription process.
 Positive Regulation
 For transcription to continue, a positive transcription
elongation factor (kinase) is needed to move forward.
 Kinase will phosphorylate both NELF & DSIF
 It will dissociate NELF
 This is a vital occurrence, for elongation to commence
BIOCHEMISTRY
Figure 17. Regulation of mRNA synthesis. NELF: Negative Elongation
Factor; DSF: DRB-sensitivity inducing Factor
IV. TRANSCRIPTIONAL MODIFICATIONS
A. CO-TRANSCRIPTIONAL MODIFICATIONS
 As transcription occurs, modifications on the primary transcript
also occur.
 While in prokaryotes the base sequence of DNA is transcribed
into an mRNA that is immediately and directly translated into the
amino acid, eukaryotic RNA transcripts require significant
alteration before they are transported to the cytoplasm and
translated.
Capping
 Modification occurring at the 5’ end.
 A 7-methylguanosine (7-mG) cap is added to the 5’ end
 The cap protects the 5’ end of the molecule from nuclease
attack.
o Prevents premature degradation
 The cap is fairly complex and is distinguished by the unique
5’-5’ bonding that connects it to the initial ribonucleotide of
the RNA. Some eukaryotes also acquire a methyl group
(CH3) at the 2’-carbon of the ribose sugars of the first two
ribonucleotides of the RNA.
o Chemical bond formed: 5’-5’ phosphodiesterbond
between the 7-methylguanosine cap and the first
ribonucleotide in the RNA sequence
o Source of cap: GTP
o Cap 0: The guanosine in GTP is methylated by methyl
transferase. After which, it is added to the 5’ end of
mRNA via the capping enzyme
 A capping enzyme is responsible for adding GTP to the 5’-
end of mRNA. The guanine of cap is methylated at 7-
position
 For the addition of methyl group at the ribose sugars of the
first two ribonucleotides in the RNA sequence:
o Methyl donor: Adenosylmethionine (SAM)
o Enzyme: Methyl transferase
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o Cap 1: first nucleotide of the transcript is methylated at
C2’
o Cap 2: second nucleotide of the transcript that is
methylated at C2’
Figure 18. Formation of the 5’-5’ phosphodiester bond between the 7-
methylguanosine cap and the first ribonucleotide in the RNA sequence
signal (AAUAAA), a CA dinucleotide a few bases
downstream, and a GU-rich tract
2) The polyadenylation complex consists of several proteins
that bind to these sequences, including poly(A) polymerase
itself, cleavage factor and poly(A)-binding protein (PABP)
3) The growing RNA is cut just beyond the CA site by cleavage
factor
 There is cleavage between the CA and GU-rich
sequences. Therefore, GU-rich tract will be lost and
the CA dinucleotide will remain
 After this cleavage, poly (A) polymerase attaches
adenine residues to the CA dinucleotide. This
would be the start of the synthesis of the poly-A tail
4) Poly(A) polymerase adds the poly(A) tail to the free 3’-end of
the mRNA. The completed poly(A) tail is bound by PABP
(Clark)
 The rest of the polyadenylation complex would
dissociate except for the PABP

Figure 20. mRNA sequence showing the sequences beyond the end of
the coding sequence, which are involved in cutting and tail addition:
AAUAAA, CA dinucleotide, GU-rich

Figure 19. Capping of mRNA

Formation of the Poly (A) Tail

 Modification at the 3’- end.
 A polyadenylation signal contains an AAUAAA sequence.
 AAUAAA polyadenylation signal- signals that the
poly (A) tail must be produced at the 3’ end
 There is usually a GC rich region in the primary mRNA where
the polyadenylation enzyme complex attaches. The
polyadenylation enzyme complex contains:
 CST: pairs with GU-rich sequence
 Poly A polymerase: associates with the CA
dinucleotide
 Poly A Binding Protein (PABP): binds to the tail
after its synthesis; important for translation
 Cleavage Polyadenylation Stimulating Factor
(CPSF): must identify and associate itself with the
polyadenylation signal (AAUAAA)
 Process of the addition of Poly (A) tail:
1) During transcription RNA polymerase continues on beyond
the end of the coding sequence until the RNA is cut free.
Figure 21. Addition of PolyA tail to eukaryotic mRNA
Three important sequences beyond the end of the coding
sequence are involved in cutting and tail addition: the tail

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Splicing: Removal of introns
 Primary transcript contains introns, which have to be  The spliceosome consists of several ribonucleoproteins (U1
removed to U6), known as “snurps”, which are involved in splicing.
These assemble at the splice sites at the intron/exon
 Introns: non-coding sequences; sequences that
boundaries (Clark).
are not represented in the final mRNA product (“int”
for intervening)
 Exons are joined together, eliminating introns from the
primary transcript and forming a continuous sequence that
specifies a functional polypeptide.
 Exons: coding sequences; sequences that are
retained to code for the protein products
 Splicing involves the removal of the corresponding introns
and rejoining of the exons
 Enzyme: Small nuclear ribonucleoproteins (snRNPs)
 5’ end and 3’ end of exons are joined by the by the 5’ end of
the intron, forming an AGGU nucleotide sequence.
 Spliceosomes are required to recognize the AGGU sequence
so that successful removal of the intron will occur.
 Spliceosome/SNRNPS: responsible for splicing

Figure 22. Sequence at the splice junctions

 Lariat Formation: Successful splicing process requires base

pairing; there should be successful recognition of the 5’ end
of the intron to be spliced.
 In mammalian cells, this consensus sequence is
PyNPyPyPuAPy, where Py is a pyrimidine, Pu is a
purine, and N is any nucleotide. The branch site is
located 20–30 nucleotides upstream from the 3' site
(Harpers)
 First, the intron and exon are cut apart at the 5’ Figure 25. Stages in the assembly of spliceosomes
splice site and the free 5’-end of the intron loops
around and is joined to the adenine at the branch 1.) U1 binds the 5’ splice site and U2AF binds the 3’ splice site
site. 2.) U2 binds the branch site
 Second, the free 3’-end of the upstream exon 3.) U4 and U6 bind to U2
displaces the intron from the 3’ splice site and the 4.) U5 binds to the downstream exon
two exons are joined together. The intron is 5.) A loop forms because of the association of U1 and U2
released as a branched lariat structure. The 6.) U6 displaces U1 from the spliceosome and U4 departs as
released introns will give rise to “guide RNAs” well (Clark).
Take note that there is no U3 – Doc Magat

Clinical Correlation: B-thalassemia

 One of the most common causes of hemolytic anemia.
 RBC becomes fragile and brittle
 B-globin chain is defective due to the abnormal sequence of
exon-intron boundaries.
 RBCs are pale-looking, with loss of biconcave shape.
 RBCs have shorter half-life
 Defects can occur during splicing or addition of poly (A) tail
 Not all are spliced producing abnormal proteins
 Formation of an abnormal polyadenylation signal, rather than
AAUAAA, AACAAA is formed

Figure 23. The splicing reactions. Take note of the “lariat formation”
(like a loop) that occurs during splicing
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Figure 26. Summary of Co-transcriptional modifications in eukaryotic mRNA
B. POST TRANSCRIPTIONAL MODIFICATIONS
 Occurs after the transcription of the mRNA molecule.
 In eukaryotes, transcription happens in the nucleus while
translation occurs in the cytoplasm, where ribosomes are
located
 Thus, transcribed RNA has to be moved outside the
nucleus for it to be translated
 These post transcriptional modifications occur after
the mRNA molecule has appeared in the cytoplasm
or as it goes to the cytoplasm
 Secondary methylation:
 Methylation of nucleotides in the mature mRNA
specifically on the 2’OH and at N7 of adenylyl
residues.
 5’ UTRs and 3’ UTRS - specific relevance in the regulation of
translation
 functions of 5’ UTRs and 3’ UTRs are implicated in
RNA processing, transport, degradation, and
translation; each of these reactions potentially
contribute to additional levels of control of gene
expression
 The different post-transcriptional modifications are done to
prevent premature decay of mature mRNA
TERMINATION
 Once the RNA transcript is completed, transcription is
terminated
 When TFIIH detaches itself, the RNAP II is
dephosphorylated and recycled, ready to initiate another
transcript
VI. rRNA SYNTHESIS
 The process of transcription discussed above is for the
transcription of messenger RNA (mRNA). It is important to note
that there are different types of RNA and they are synthesized
in different ways.
 rRNA fabricates the polypeptides and provides a mechanism for
decoding mRNA into amino acids and interacts with the tRNA
during translation. It is the most abundant type of RNA (about
80%) in the cell.
 Facilitated by RNA polymerase I (RNAP I) and synthesis occurs
in the nucleolus
 Eukaryotes contain many copies of the genes for ribosomal
RNA. These are found in clusters and are transcribed by RNA
Polymerase I. (Clark)
BIOCHEMISTRY
 Overview:
 The genes for rRNA are located at multiple sites along the
DNA. A single transcribed unit of DNA yields an initial RNA
molecule of 45S. The 45S RNA is processed to yield the final
18S and 28S subunits of rRNA.
 In eukaryotes, there are 4 rRNAs: the 5S rRNA is made
separately and does not need processing. The other three
(18S rRNA, 28S Rrna, and 5.8S rRNA) are made as a single
pre-rRNA.
Figure 27. Clusters of rRNA genes
A. INITIATION
 Molecules of rRNA are synthesized in the nucleolus.
 Core promoter and upstream control element: GC rich
regions (take note! Different from the promoter sequence found
in promoter regions for mRNA transcription)
 This promoter region will be recognized by Upstream
Binding Factor 1 (UBF1) to allow for the bindning of
RNA polymerase
 There are less upstream promoter elements in rRNA synthesis.
 The core promoter is such because specific transcription factors
are required.
 Transcription of rRNA can happen simultaneously, unlike
mRNA, where transcription, co-transcriptional modification, and
cleaving result to the production of just one mRNA after the
other.
 Steps leading up to initiation:
 UBF1 binds to distinct regions in the coding region.
 After the binding of UBF1, Selectivity factor 1 (SL1), made of
four polypeptides including TBP (TATA Binding Protein)
binds as well.
 UBF1 and SL1 are both required to assemble and attach
themselves to the core promoter region before rRNA
transcription will occur
 Once UBF1 and SL1 are in place, RNAP I can now bind with
the help of TIFIA (also called Rrn3).
 Assembly of the initiation complex then proceeds to rRNA
transcription.
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Figure 29. rRNA: Post-transcriptional modification.

There is a pre-rRNA 35S and 41S. After cleavage and degradation of the
linker regions, you are left with mature rRNA: 20S rRNA, 5.8S rRNA, and
28S rRNA

C. FORMATION OF BASE MODIFICATION

 In eukaryotic rRNA, modification occurs at multiple sites and
requires small RNA guide molecules called guide RNA (gRNA)
in addition to modification enzymes.
 gRNA locate the correct sites for modification by base pairing
(usually G-U) over a short region with the rRNA.
 Also known as small nucleolar RNA (snoRNAs) since
synthesis and processing of rRNA in eukaryotes occurs in
the nucleolus
Figure 28. RNAP I transcribes rRNA genes.  snoRNA are guide ribonucleoproteins, not snRNPs,
The promoter or RNA polymerase I has an upstream control element and a
which are spliceosomes. Spliceosomes remove introns
core promoter, the latter rich in CG sequences. The UBF1 protein
recognizes and binds to both the upstream control element and the core from a transcribed pre-mRNA (this distinction was
promoter. Subsequently, SL1 binds to the DNA in association with UBF1. stressed by Dr. Magat)
Finally, RNA polymerase I binds and transcription commences.
Note: Remember that guide RNAs (gRNA) / snoRNA are
B. POST-TRANSCRIPTIONAL MODIFICATION formed from the introns obtained from splicing process. The
 In rRNA, the primary transcript needs to be formed first before snoRNA is made by cutting up the intron after it has been
the modifications. spliced out of the mRNA.
 The primary transcript would have the highest and heaviest
sedimentation rate because it contains the parts to be retained,
mature rRNA, and the proteins described as the linker regions.
The linker regions would have to be cleaved out to produce
mature rRNA.
 Introns are not present. Instead, spacer regions are
present which would be cleaved off to produce
different sizes of rRNA
 Series of cleavage in the 45S transcript will occur in order to
produce mature rRNAs

Figure 30. Recognition of modification sites on rRNA by snoRNA. Take note

of the base paring between snoRNA and rRNA sequence. This base pairing
usually occurs in the GU rich sequence of the rRNA

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 The site on RRNA to be modified is identified by base pairing to
specific sequence on the snoRNA. After snoRNA/rRNA pairing,
the methylase binds to the BoxC and BoxD sequences and
then methylates one of the bases of the rRNA
 Formation of snoRNA:
 After splicing the mRNA, the intron assumes a lariat
structure; snoRNA is spliced from the intron, leaving
smaller fragments of RNA.
Figure 31. Generation of snoRNA from Intron.
 Nucleotides in eukaryotic rRNA are modified by methylation
 Methylation of the ring nitrogen of the base on rRNA,
usually adenine
 Methylation of the 2’-OH group of the ribose of the
45S precursor.
o Methyl groups may serve as markers for
cleavage of the 45S precursors and are
conserved in the mature rRNA.
Figure 32. Maturation of the 45S rRNA precursor.
The clear regions are removed, and the red regions become the mature
rRNAs (The 5S rRNA is not produced from this precursor).
 Another base modification is the conversion of uridine to
pseudorudine (pseudouridylation):
 the double bond within the uracil (between 5 and 6) is
reduced. Hence the term, pseudouridine. This is similar to
what happens in tRNA.
D. SUMMARY OF rRNA SYNTHESIS
BIOCHEMISTRY
Figure 33. Summary of rRNA synthesis.
The formation of rRNA occurs in the nucleolus. First, the primary transcript
is formed. Methylation, trimming and removal of the linker regions to
produce the different types of rRNA then occur. Export to cytoplasm of the
40S ribosomal subunit and the 60 S ribosomal subunit would form the 80S
ribosome for the process of translation (Magat).
 The 5S rRNA is transcribed in the nucleoplasm and moves into
the nucleolus.
 The other rRNAs are transcribed from DNA and mature in the
nucleolus, forming the 40S and 60S ribosomal subunits, which
migrate to the cytoplasm (Mark’s).
VII. tRNA SYNTHESIS
 Mature cytosolic tRNAs, which average 75 – 80 nucleotides in
length, are produced from larger precursors (pre-tRNAs)
synthesized by RNA polymerase III in the nucleoplasm
 Mature tRNAs also contain numerous modified bases that are
not present in tRNA primary transcripts.
 A tRNA has one binding site for a specific sequence of 3
nucleotides in mRNA (the anticodon site) and another binding
site for the encoded amino acid (Mark’s)
 Ensure that the genetic code is translated into the correct
sequence of amino acids.
A. SECONDARY STRUCTURE OF tRNA
 A tRNA molecule has a distinctive folded structure with three
hairpin loops that from the shape of a three-leafed clover.
 Structure:
 D loop: loops closest to the 5’end which contains
dihydrouridine (D)
 Anticodon loop: contains the trinucleotide anticodon
that base-pairs with the condon on mRNA
 TΨC loop: contains Ribothymidine (T) and
Pseudouridine
 Variable loop: varies in size and is found between the
anticodon loop and the TΨC loop
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Figure 34. Structure of tRNA
B. INITIATION
Figure 35. Internal promoter for RNAP III.
The gene for 5S rRNA is transcribed using a promoter located within the
gene itself. The recognition sites are downstream of the start site. TFIIIC (τA
and τB subunits) (or TFIIIA) binds to both sites and this induces TFIIIB
(Brf1, Bdp1 and TBP subunits) to bind to the promoter near the start site.
Only after TFIIIB binds can RNA polymerase III bind (Clark).
 Unique to tRNA promoter regions would be the blocks: A block
and B block downstream
 The promoter regions are present downstream, which
means that the promoter regions are present in the
genes transcribed – referred to as Internal control
genes
 This differs from the promoter regions for the
transcription of mRNA and rRNA, which are found
upstream before the genes to be transcribed
 Transcription requires the binding of either of two proteins
known as TFIIIA and TFIIIC (each with 2 subunits: tA and tB) to
a region over 50bp downstream from the start site (Clark).
 Once these have bound, the complex will call on TFIIIB, which
is composed of 3 polypeptides:
 Brf1
 Bdp1
 TATA Binding Protein (TBP)
 Only then will RNAB III attach to the promoter region and so the
production of tRNA can happen.
BIOCHEMISTRY
B. POST-TRANSCRIPTIONAL MODIFICATION
Timming
 The primary transcript of the tRNA gene contains extra
nucleotide sequences in both the 5’ and 3’ of the tRNA
sequence
 The primary transcript is trimmed to yield a precursor molecule
with shorter 5’ and 3’ extensions
 Ribonuclease E or F – cleaves the precursor RNA near
the 3’ end
 Ribonuclease D – chews off bases from the new 3’ end,
leaving CCA at the end of the acceptor stem
 Ribonuclease P – cleaves off the 5’ end precisely
 To close the opening, a 2’ and 3’-phosphate group from one
end is ligated to 5’ hydroxyl, on the other end by an RNA ligase
Figure 36. Processing of tRNA.
Nucleotides shown in red are removed. First (1) ribonuclease E or F
cleaves the precursor RNA near the 3’ end. Second (2), ribonuclease D
chews off bases from the new 3’ end leaving the CCA at the end of the
acceptor stem. Third (3), ribonuclease P cleaves the 5’ end precisely
(Clark).
Base Modification
 tRNA has a clover-leaf structure that folds into a 3-D L shape
and contain several bases that are modified
posttranscriptionally.
 The bases are modified at the same time the endonucleolytic
cleavage reactions are occurring (Mark’s).
 The modification of bases in tRNA would include (Magat):
 Pseudouridine at the D loop
 Bond between carbons 5 and 6 is reduced hence the term
pseudouridine.
 Uracil is rotated, forming a 5 and 1’ O-glycosidic bond,
present in the T Ψ C loop.
 Uracil is methylated by SAM, forming ribothymidine.
 Deamination of adenine forms hypoxanthine.
 Dihydrouracil is formed through the reduction of the double
bonds of uracil.
 The final step in forming the mature mRNA is the addition of a
CCA sequence at its 3’-end. These nucleotides are added one
at a time by tRNA nucleotidyltransferase (Mark’s).
 Nucleotidyltransferase uses ATP and CTP and
substrates and always incorporates them into tRNA at a
ratio of 2C/1A
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Figure 37. Modification of Bases.
The tRNA cloverleaf. Bases that commonly occur in a particular position are
indicated by letters. Base-pairing in stem regions is indicated by lines
between the strands. The locations of the modified bases dihydrouridine
(D), ribothymidine (T), and pseudouridine (Ψ) are indicated (Mark’s).
Splicing
 In some cases, primary transcripts may contain introns in the
anticodon region of the tRNA
 Intron removal is dicated by the secondary structure of the
precursor and is carried out by a soluble, two component
enzyme system; one enzyme removes the intron and the other
reseals the nucleotide chain.
C. SUMMARY OF tRNA SYNTHESIS
Aminoacyl-tRNA synthetase
Figure 38. tRNA Synthesis.
The primary transcript is formed. Trimming and phosphorylation at the 5’
and at the 3’ end occur. Attachment of CCA acceptor stem at 3’ end
happens next. Trimming and ligation of the introns on the anticodon loop
then occur. The bases are then modified (Magat).
Ribonucleoprotein Particles (RNP Particles)
 RNA polymerase III is the enzyme that produces tRNA. The
promoter is located within the coding region of the gene
 Primary transcripts for tRNA are cleaved at the 5’ and 3’ends
 Some precursors contain introns that are removed
 During processing of tRNA precursors, nucleotides are modified
 Post transcriptional modification includes the conversion of
uridine to pseudouridine (Ψ), ribothymidine (T), and
dihydrouridine (D)
 Addition of the sequence CCA to the 3’ end is catalyzed by
nucleotidyl transferase
BIOCHEMISTRY
VIII. EXPORT TO CYTOPLASM
 The formation of RNA in the nucleus should involve export to
the cytoplasm.
 mRNA, tRNA, and rRNA function in the cytoplams. They move
out of the nucleus through the nuclear pore.
A. NUCLEAR PORE
Figure 39. Nuclear pore.
Each nuclear pore is surrounded by a cluster of proteins that control entry
and exit.
 The export to the cytoplasm is through the nuclear pore.
However, the process is not just extruding the RNA into the
cytoplasm. It is a complex process.
 The nucleus is surrounded by a double membrane and has
many pores that allow macromolecules in or out in a carefully
controlled manner (Clarks)
 Exportins and importins are the protein factors known to control
the exit and entry of specific classes of molecules through the
nuclear pores.
 Exportin-t – specific for export of tRNA
 Exportin 5 – for microRNA precursors
 Transport out of the nucleus or large molecules (like RNA and
Proteins) require energy via the hydrolysis of GTP
 Once the mRNA has received its cap and tail and had its introns
spliced out, it is free to exit the nucleus.
B. ENZYMES AND MOLECULES
 RNAs are too big to pass thru the nuclear pore thus for export to
occur, they have to be bound to proteins to form
ribonucleoprotein (RNP) particles
 For successful export to cytoplasm, RNAs should be associated
with RNP, specifically mRNA, which has a very short half-life.
 Even if it’s normal, biologically functional mRNA, it can
be easily degraded by a lot of ribonucleases.
 Important in translation, associated with export to the cytoplasm
 Catalyzes the activation of amino acids by linking them to their
appropriate tRNA carriers so they can be incorporated into
proteins
 Has a role in the formation of P bodies
 Abnormal, easily degraded RNA can be screened by RNP
particles. This occurs in the P bodies.
 Form of mRNAs bound by specific packaging proteins for export
from the nucleus to the ribosomes for translation (protein
synthesis)
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P Bodies
 Cytoplasmic organelles that are sites of translation repression
and mRNA decay
 Formed by association of specific mRNAs with protein
constituents.
 Some defective RNAs are processed here for degradation. It’s
not only for export to the cytoplasm but also for further
screening. If the RNAs are defective, they will be degraded.
IX. DECAY OF NORMAL EUKARYOTIC RNA
A. RNA DECAY
Figure 40. Steps in RNA Decay
 RNA is removed from the cytoplasm by cellular
ribonucleases. mRNAs are initially degraded in the
cytoplasm and the rates are varied depending on the mRNA
species thus raising the possibility of control by differential
degradation.
 There are different kinds of nucleases but the most important
are exonucleases and endonucleases:
 Exonucleases - degrade RNA either from the 5’ – or 3’ –
end
 Endonucleases - cleaves phosphodiester bonds within a
molecule (inside the sequence)
 Most commonly, the 3’ poly (A) tail of mRNA is shortened
primarily by exonuclease digestion.
 Deadenylation is inhibited by poly (A)-binding proteins
(PABP). Then the cap is removed by a specialized
decapping enzyme, which is also inhibited by poly (A)-
binding protein.
 A decapped RNA is the substrate for a 5’-3’ exonuclease that
degrades the RNA to nucleotides.
 An alternative pathway involves deadenylation and 3’-5’
exonucleolytic degradation by cytoplasmic exosomes of
mRNAs that still retain their cap (Devlin).
Steps in mRNA decay
1. The polyA tail of mRNA is shortened by digestion via
exonucleases
 Initially, there is a decay on the 3’ end
 Inhibited by poly(A)-binding protein (PABP)
 Once the Poly (A) tail is shorterned to 10-20 bases, PABP is
released
2. Specialized decapping enzymes remove the cap
 Can digest the cap at the 5’ end
 Inhibited by poly(A)-binding protein
BIOCHEMISTRY
3. 5’ – 3’ exonuclease degrades the decapped RNA into
nucleotides
 Superkiller can digest 3’ to 5’ direction
Alternatively:
 Polynucleotide phosphorylase degrades RNA via 3’ – 5’
exonuclease activity.
 Inorganic phosphate is used to displace the phosphodiester
bondat the 3’-end of RNA, yielding a shorter RNA and a
nucleotide diphosphate
B. NONSENSE MEDIATED RNA DECAY
 Occurs in P Bodies
 Associated with abnormal mRNA and to some extent,
normal mRNA.
 Eliminates mRNAs incapable of making complete proteins.
 A misprocessed or mistranscribed RNA would have a
high probability of carrying chain-terminating (nonsense)
codons
o A presence of a nonsense codon causes
premature termination
 Premature nonsense codons trigger mRNA destruction, a
process termed nonsense-mediated decay.
 During the first round of translation, any premature
termination codon is recognized by termination factors.
 This complex recruits other factors, which signal mRNA
destruction and send the defective mRNA to the P body.
 This surveillance mechanism is able to distinguish
between premature and correct nonsense codons
because the former occur upstream (5’ to) intron-exon
junctions of the mRNA.
In nonsense-mediated decay, the first step is removal of the cap
from the mRNA. (This contrasts with normal mRNA degradation
where the poly (A) tail must be removed before the cap). Next, the
mRNA is degraded from the exposed 5’- end. (Clark)
IX. REGULATION
A. EPIGENETIC MECHANISM OR CHROMATIN
REMODELING
 Remember that DNA are wound around histones to form
nucleosomes
 Histones allow compression of the DNA molecule into
nucleosomes to create the densely packed chromatin
 However, it can be a problem when transcription factors &
polymerases are unable to bind to the DNA as a result of
the tight packing
 Heterochromatin – densely packed DNA; cannot be
transcribed
 Euchromatin – loosely packed DNA
 An ionic bond is present between the DNA and histones
 Histones are have with the amino acid Lysine, which is
positively charged
 DNA generally possesses a negative charge; thus ionic
bonding is present between the two
 In regulation, covalent bonding exists not only on the primary
DNA structure, but also on other structures such as the
histones
 Histones need to be regulated via covalent modification
specifically in their N-terminal regions which are projecting
outward and are exposed to the nuclear surface
 Interactions due to the said tails/region are important in
nucleosome aggregation & higher-level folding of chromatin
 Covalent histone modifications include:
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 Aggregation Aggregation and Disaggregation
 Disaggregation
 Methylation (H3)
 Acetylation (H4)
 Phosphorylation
 Ubiquination

Histone Acetyl Transferase (HAT)

 Acetylation of histone tails is performed by the co-activators
known as Histone Acetyl Transferase (HATs)
 Note from Doc Magat: acetylation is from the Acetyl CoA
molecule, which is from the PDH complex (fed state) that has
glucose as a substrate. Thus, the process of transcription is
facilitated by the fed state
 Histone acetyltransferase – attaches acetyl groups to the
basic and positively charged histones, specifically to the lysine Figure 42. Acetylation of histone tails disaggregates the nucleosomes
residues
 Acetylated lysine residues weaken the positive charge,  Occurs in the nucleus
therefore also weakening the bond between DNA and  Closely packed nucleosomes are stabilized by the binding of the
histones histone tails of one nucleosome to the next nucleosome
 Leading to unwinding of the DNA around the histone
 Aggregated nucleosome results to gene repression
 Unwinding would cause disaggregation and exposure of  Important histones to note: H3 & H4 – Doc Magat
loop DNA
 When the H4 tail is acetylated, it no longer binds to histones in
 Goal: expose promoter site and the gene, to be more
an adjacent nucleosome
accessible
 Promoting disaggregation of neighboring nucleosomes
 The degree of acetylation affects:
 Disaggregated = enhanced promoter access (due to loose
 State of nucleosome aggregation
structure)
 And therefore, gene expression
 The lysine residues binding one nucleosome to the other make
 Non-acetylated histone: form highly condensed
them aggregate very closely
heterochromatin
 Acetylation of the lysine residues disaggregates the
 Acetylated histone: less condensed chromatin
structures further apart
 Deacetylation on the other hand is due to a repressor complex
 Acetyl CoA comes from citrate and uses citrate lyase (fed
that combines both a DNA-binding subunit and a deacetylase
state), reiterating the idea that transcription is a process
 This would prevent diaggregation and therefore would inhibit
facilitated by the fed state
transcription
 Aside from acetylation, there are actually other several covalent
modifications for histones such as Methylation
 Methylation of H3 on Lys residues 4 & 36 = enhanced
gene expression
 Methylation of H3 on Lys residues 9 & 27 = gene
repression

Histone Modification/Remodeling
 Mainly involves unwinding of DNA around histones
 Facilitated by:
 SWI/SNF (switch sniff)
 Can both slide and remodel nucleosome
 ISWI (imitation switch)
 Can only slide nucleosome and cannot rearrange them;
bind to histones rather than DNA
 Sliding – slide nucleosomes along DNA molecule to expose the
transcription sequences
Figure 41. Acetylation and deacetylation of histones  Exposes a previously inaccessible promoter
 Remodeling – rearranging histones, remodeling nucleosomes
Note: into looser structure for easier DNA access; uses ATP as
 In histone modification, the lysine residues in energy source
histones are acetylated, NOT THE DNA!  Can merge 2 nucleosomes to loosen DNA winding, making
 mRNA coding for histones do not have poly-A tail at more of the DNA accessible
their 3’ end
Use of Remodeling Complexes
 Histone complexes 1 and 2 are affected
 Promotes the release of DNA from the histones
 Generating access to more promoter regions by the
transcription complex

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Figure 45. Transcriptional control of HMG CoA by SREBP1

Figure 43. Sliding and remodeling of nucleosomes
 HMG CoA Reductase is the rate limiting enzyme for the
synthesis of cholesterol, which is normally active during the
fed-state
 HMG-R transcription is controlled by the transcription factors
known as SREBPs (sterol regulatory element binding
proteins)
 An increase in HMG CoA leads to increase in cholesterol de
novo synthesis and LDL-R synthesis

Step-by-step regulation of HMG CoA

1. SREBP1 is bound to SREBP cleavage activating
protein (SCAP) in the ER membrane
2. When cholesterol levels drop, creating a need to
increase cholesterol synthesis, SREBP:SCAP
complex is translocated to the Golgi apparatus.
3. In the golgi apparatus, proteolytic cleavage at sites 1
& 2 via S1P and S2P proteases, respectively,
releases DNA binding domain of SREBP (N-terminal)
from golgi membrane
 SREBP becomes biologically active; translocates to nucleus
from cytosol to look for the Steroid response element (SRE)
and promote gene transcription.

LDL-Receptor Synthesis:

Figure 44. Proposed mechanism of SWI/SNF nucleosome eviction

B. REGULATION OF HMG COA REDUCTASE

 There are numerous processes by/through which gene
expression is regulated at the transcription step:
 Availability of GSTF
 Chromatin remodeling
 Alternative promoter sites Figure 46. Transcriptional regulation of LDL-R. SRE binding protein,
GC=GC rich region, and activators present upstream.
 Present for the different isoforms of key enzymes
 RNA Editing
 Once SREBP1 is bound to SRE, it requires Specificity Protein 1
(SP1) , which attaches to the GC-rich region found upstream of
the promoter site
 DNA will wind itself to predominantly basic histones and
proteins
 CRSP (co-factor required for Sp1)
 CBP (cAMP response element binding protein) has the histone
acetyl transferase function for chromatin remodeling

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Gene Expression II: Transcription
 Certain molecules that undergo covalent modification,
specifically acetylation, enhances the LDL-R and cholesterol
synthesis

Glucokinase
 Regulation via the presence of alternative promotion sites
 Differential regulation of the glucokinase gene is accomplished
by using tissue specific promoters as it has different isoforms
 Glucokinase (hexokinase 4) have different isoforms
 The isoform depends on the exposed promoter region of the
gene to be transcribed

Figure 49. RNA editing of Apo B48 & B100

Figure 47. Glucokinase promoter sites in differenct cell types
Figure 48. Glucokinase Regulation
Apo B48 & B100
 Regulation occurs via tissue specific RNA editing
 Apo B48 – in the intestine = Chylomicrons
 Intestinal mRNA altered by base editing to make Apo B48
 Apo B100 – in the liver = VLDL
 In the intestines, CAA sequence is targeted to become UAA
(stop codon)
 A deaminase binds to the CAA region on the mRNA,
cytosine has an amino group on position 4, which will be
removed converting the cytosine nucleotide to uracil
 This causes mRNA translation to terminate earlier in the
intestine
 However in the liver, there is complete transcription which
results to the translation of Apo B100
PEPCK/G6Pase Synthesis
 Phosphoenol pyruvate carboxykinase (PEPCK) and Glucose-6-
phosphatase (G6Pase): for gluconeogenesis
 Many GSTFs exist in the promoter region and the regulation
occurs via the presence of the different transcription factors
 PEPCK is an “emergency enzyme” because it sustains normal
blood glucose levels in stressful states such as fasting
 Critical component of normal brain function and survival
through fight or flight responses
 Transcriptional control through mTORC2 – formerly mammalia
TORC2, now mechanistic TORC 2 – in T2DM patients with
uncontrolled hepatic gluconeogenesis
 mTORC2 becomes very active in uncontrolled DM type 2
 It will induce the translocation of cAMP in the nucleus
 cAMP binds to CREB
 This complex attaches itself to the promoter region,
attracting mTORC
 mTORC attracts PGC1 α, inducing the successful
transcription and translation of mRNA producing PEPCK
and G6Pase
 Leads to the uncontrolled mRNA production to produce
PEPCK/G6Pase
 This would ultimately result to the uncontrolled hepatic
gluceoneogenesis, which increases the glucose levels in the
blood
Figure 50. Transcriptional Control of PEPCK synthesis

 In the diagram above, some of the known transcription factors

that regulate transcription of the PEPCK gene are depicted
 Expression of the gene depends on the combined input of all
the transcription factors present (reflecting the current state of
the cell)

X. CLINICAL CORRELATION: METFORMIN

A. MECHANISM OF ACTION

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TiTa
Gene Expression II: Transcription
 Metformin is a drug used to treat T2DM for more than 30 years c. Lys residue 4
 An antihyperglycemic agent which improves glucose d. Both A and B
tolerance by lowering basal and postprandial blood glucose e. Both B and C
 Decreases hepatic glucose production
 Decreases intestinal absorption of glucose 4. The sigma σ subunit of prokaryotic RNA polymerase is
a. Part of the core enzyme
 Improves insulin sensitivity by increasing peripheral glucose
b. Inhibited by antibiotic rifampicin
uptake/utilization
c. Must be present for transcription to occur
 Although insulin secretion remains the same, fasting insulin
d. Specifically recognizes promoter sites.
levels and day long plasma insulin response may decrease
5. Which which ribonucleoprotein replaces U1 in the final
 TORC2, under normal conditions, works with CREB, which is stage of spliceosome assembly?
activated by increased level of cAMP a. U2
 Stimulate increased transcription of genes required for b. U3
gluconeogenesis c. U3AF
d. U6
 Metformin stimulates the activation of AMPK, which
phosphorylates TORC2 and sequesters it in the cytoplasm. This Answers: 1FALSE (enhanced promoter access), 2FALSE (upstream) 3E, 4D, 5D
decreases the synthesis of gluconeogenic enzymes and
reducing hepatic output of glucose REFERENCES
 Phosphorylation of TORC2 deactivates the enzyme
and this decrease gluconeogenesis 2020AC Trans
 Action of metformin in decreasing glucose levels Lecturer’s PPT/Study Guide

MOA in Glucose Metabolism Devlin T.M. Textbook of Biochemistry, 4th ed.

 Metformin upregulates formation of liver kinase B1 Baynes J.W., Dominizack M.H. Medical Biochemistry, 4th ed.
 LKB1 enhances AMPK activity Lieberman M., Marks A.D., Smith C. Marks’ Basic Medical
 AMPK phosphorylates TORC2 Biochemistry 2nd ed.
 TORC2 is prevented from translocating to the nucleus
 PEPCK and G6Pase production are not successful
 Gluconeogenesis is not stimulated
 Blood glucose levels are controlled

MOA in Lipogenesis
 Metformin inhibits SREBP1
 Further inhibiting key enzymes in lipogenesis through
transcriptional control
 HMG-R
 FA synthase
 Acetyl CoA carboxylase
 Key enzymes controlled though phosphorylation:
 HMG-R
 Glycerol-3-phosphate acetyltransferase
 Acetyl CoA carboxylase
 Malonyl CoA decarboxylase
 Only one activated while the former 3 are inhibited
 Malonyl CoA decarboxylase enhances B-oxidation of FAs in
skeletal muscle. Instead of synthesis of lipids which happens
when SREBP1 is activated, degradation/B-oxidation takes
place.
 Both covalent modification and transcriptional control on HMG-
R and Acetyl CoA carboxylase are exhibited by metformin
 Metformin decreases FA and cholesterol synthesis, and
increases B-oxidation

REVIEW QUESTIONS
1. TRUE or FALSE: Disaggregation leads to diminished
promoter access.

2. TRUE OR FALSE: TFIIA binds downstream the TATA box

and guides RNA Pol II in place

3. Covalent modification enhances gene expression

through methylation of which H3 lysine residues?
a. Lys residue 9
b. Lys residue 36

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