RNA Structure,

Chapter 3:
Synthesis, and
Processing
Course Coordinator: Dr. Arika, Ph.D
Course Overview:
Goals:
To learn the basic mechanisms of
transcription, RNA processing and
translation (protein synthesis).

Course content include:

1. The Structure of RNA
2. Transcription of Protein-coding Genes
and Formation of Functional mRNA
3. Decoding of mRNA by tRNAs
4. Stepwise Synthesis of Proteins on
Ribosomes
RNA (Ribonucleic acid)
• The genetic master plan of an organism is contained in
the sequence of deoxyribonucleotides in its
deoxyribonucleic acid (DNA). However, it is through the
ribonucleic acid (RNA)— the “working copies” of the DNA
—that the master plan is expressed

• The copying process, during which a DNA strand serves as

a template for the synthesis of RNA, is called
transcription.

• Transcription produces messenger RNAs that are

translated into sequences of amino acids (polypeptide
chains or proteins), and ribosomal RNAs, transfer
RNAs, and additional small RNA molecules that
perform specialized structural, catalytic, and regulatory
functions and are not translated.

• The final product of gene expression, therefore, can

be RNA or protein, depending upon the gene.
Structure of RNA
There are three major types of RNA that participate in the process of protein
synthesis:
1- Ribosomal RNA (rRNA)
2- Messenger RNA (mRNA)
3- Transfer RNA (tRNA)

• Like DNA, these three types of RNA are unbranched polymeric molecules
composed of nucleoside monophosphates joined together by
phosphodiester bonds.

• They differ as a group from DNA in several ways:

• They are smaller than DNA
• They contain ribose instead of deoxyribose
• They contain Uracil instead of thymine.
• Most RNAs exist as single strands that are capable of folding into
complex structures.
 1- Ribosomal RNA (rRNA): make up 80
% of total RNA in the cells are rRNA.

• rRNA are found in combination with several

proteins (about 82 proteins) as component of
the ribosome. Which is the site of protein
synthesis.
• In Eucaryotic ( mammals),there are 4 size
types of rRNA (5S, 5.8S, 18Ss and 28S)
representing 2/3 particle mass of the
ribosome.
• There are three distinct size species of rRNA
(23S, 16S, and 5S) in prokaryotic cells.
• Some rRNA function as catalysts in protein
synthesis. RNA with catalytic activity is
termed a “ribozyme”.
2- Messenger RNA (mRNA):
• Comprise of only 5% of total cellular RNA.
• Function: Carry genetic information from DNA in the nucleus
to ribosomes (in cystol) where it is used as template for
protein biosynthesis.
• If the mRNA carries information from more than one gene, it
is said to be polycistronic. Polycistronic mRNA is
characteristic of prokaryotes.
• If the mRNA carries information from just one gene, it is said
to be monocistronic and is characteristic of eukaryotes.
• Prokaryotic mRNA often have several coding regions, that is,
they are polycistronic.They produce a separate species of
polypeptide.
• The addition of this 7-methylguanosine “cap”
• In contrast, each eukaryotic mRNA has only one coding permits the initiation of translation and helps
region, that is, it produces only one polypepetide chain. It is stabilize the mRNA. Eukaryotic mRNA lacking
monocistronic, the cap are not efficiently translated.
• In addition to the protein coding regions that can be
translated, mRNA contains untranslated regions at its 5 ′ -
and 3 ′ -ends. • A chain of 40–200 adenine nucleotides
attached to the 3 ′ -end.This poly-A tail is not
• Special structural characteristics of eukaryotic (but not transcribed from the DNA, but is added after
prokaryotic) mRNA include: A long sequence of adenine transcription. These tails help stabilize the
nucleotides (a “poly-A tail”) on the 3 ′ -end of the RNA chain, mRNA and facilitate their exit from the
plus A“cap” on the 5 ′ -end consisting of a molecule of 7- nucleus. After the mRNA enters the cytosol,
methylguanosine attached “backward” (5 ′→ 5 ′ ) through a the poly-A tail is gradually shortened
triphosphate linkage.
 3- Transfer RNA (tRNA):

• tRNA represents 15% of total RNA in the cell.

• Structure: 1- amino acid attachment site or amino acid acceptor: at 3´ end

which terminates with the triplet CCA.
• 2- Anticodon loop or anticodon triplet
• Functions of tRNA:
1- transport amino acids to ribosome for protein synthesis. Each tRNA carry only
one amino acid. The specific amino acid is attached enzymatically to 3' end of
tRNA.
2- recognize the specified codon on mRNA to ensure the insertion of the correct
amino acid in the growing polypeptide chain. This function is due to anticodon
triplet which binds to codon on mRNA by base pairing.
• NB: Three nucleotide bases on mRNA form a codon which is then translated
into specific amino acid.
Gene expression:
• Gene expression is the process by which information from a
gene is used in the synthesis of a functional gene product.
These products are often proteins.
• Gene expression in two major steps:
• 1-Transcription: Synthesis of mRNA from DNA. By this
process, the gene carried on DNA will transferred into RNA.
• 2-Translation: The gene on mRNA is translated into a
functional protein
• Transcription = DNA → RNA
• Translation = RNA → protein
A).Transcription: Steps in RNA synthesis in Prokaryotes:

1). Initiation:
• The transcription is initiated by the binding of RNA polymerase holoenzyme to a
specific region of DNA double helix . This site is called promoter site or promoter
region, which is not transcribed.
• The prokaryotic promoter contains characteristic consensus sequences
• a. –35 Sequence: A consensus sequence (5-TTGACA-3), centered about 35 bases to the left
of the transcription start site (see Figure 30.7)
• A base in the promoter region is assigned a negative number if it occurs prior to (to the left of,
toward the 5I -end of, or “upstream” of) the transcription start site. Therefore, the TTGACA
sequence is centered at approximately base –35.
• b. Pribnow box: The holoenzyme moves and covers a second consensus sequence (5I -
TATAAT-3I ), centered at about –10 (see Figure 30.7), which is the site of initial DNA melting
(unwinding).

Figure 30.7 Structure of the prokaryotic promoter region.

T = thymine; G = guanine; A = adenine; C = cytosine
2) RNA elongation:
• These promoter regions are recognized by sigma factor (subunit) of RNA pol
holoenzyme.
• When RNA pol holoenzyme recognizes this region, it binds to it leading to a local
unwinding (separation) of the promoter region into 2 single strands:
a- DNA strand that is transcripted into mRNA is called a template strand or
antisense strand.
b- The other strand is coding strand or sense strand that contains gene to be
translated (This strand is not transcripted, not used)
[Note: Unwinding generates supercoils in the DNA that can be relieved by DNA
topoisomerases.]

• Direction of transcription:
• RNA polymerase will read the information sequence on DNA template from 3′ → 5′
direction, so RNA is synthesized antiparallel to DNA template i.e. from 5′ → 3′
direction.
• Like DNA pol, RNA pol uses nucleoside triphosphates as substrates and releases
pyrophosphate each time a nucleoside monophosphate is added to the growing chain.
• In contrast to DNA pol, RNA pol does not require a primer and does not appear to have 3′
→ 5′ exonuclease (proofreading) activity.
Figure 30.8 Local unwinding of DNA caused by
RNA polymerase and formation of an open
initiation complex.
 3) Termination:
 The elongation of the single-stranded RNA chain continues until a
termination signal is reached. Termination can be intrinsic
(spontaneous) or dependent upon the participation of a protein
known as the ρ (rho) factor.

a. ρ-Independent termination: Seen with most prokaryotic genes,

this requires that a sequence in the DNA template generates a sequence
in the nascent (newly made) RNA that is self-complementary (Figure 30.9).
This allows the RNA to fold back on itself, forming a GC-rich stem (stabilized
by hydrogen bonds) plus a loop. This structure is known as a
“hairpin.” Additionally, just beyond the hairpin, the RNA
transcript contains a string of Us at the 3I -end.
The bonding of these Us to the complementary As of the DNA template is
weak. This facilitates the separation of the newly synthesized RNA from its
DNA template,as the double helix “zips up” behind the RNA
polymerase.

Figure 30.9 Rho-independent termination of prokaryotic transcription. A.

DNA template sequence generates a self-complementary sequence in the
nascent RNA. B. Hairpin structure formed by the RNA. “N” represents a
noncomplementary base; A = adenine, T thymine; G = guanine; C =
cytosine; U = uracil. [Note: Termination of eukaryotic transcription is not well
understood.]
b. r-Dependent
termination:
 This requires the participation of an additional protein, rho (r), which is a
hexameric ATPase with helicase activity.
 Rho binds a C-rich “rho recognition site” near the 5I -end of the nascent
RNA and, using its ATPase activity, moves along the RNA until it reaches
the RNA pol paused at the termination site.
 The ATP-dependent helicase activity of r separates the RNA-DNA hybrid
helix, causing the release of the RNA.
Clinical
Significance:
 Action of antibiotics: Some antibiotics
prevent bacterial cell growth by inhibiting RNA
synthesis. For example, rifampin
(rifampicin) inhibits transcription by binding
to the β subunit of prokaryotic RNA pol, and
preventing chain extension beyond three
nucleotides (Figure 30.10).
 Rifampin is important in the treatment of
tuberculosis.
 Dactinomycin (known to biochemists as
actinomycin D) was the first antibiotic to find
therapeutic application in tumor
chemotherapy. It binds to the DNA template
and interferes with the movement of RNA pol
along the DNA.
 Figure 30.10 Inhibition of prokaryotic RNA polymerase by
rifampin.
B). Transcription of Eukaryotic Genes

• Eukaryotic transcription involves separate polymerases for the synthesis of

rRNA, tRNA, and mRNA.
• In addition, a large number of proteins called transcription factors (TFs)
are involved.
• TFs bind to distinct sites on the DNA either within the core promoter region,
close (proximal) to it, or some distance away (distal). They are required both
for the assembly of a transcription complex at the promoter and the
determination of which genes are to be transcribed.
• [Note: Each eukaryotic RNA pol has its own promoters and TFs that bind core
promoter sequences.]
• For TFs to recognize and bind to their specific DNA sequences, the chromatin
structure in that region must be altered (relaxed) to allow access to the DNA.
Nuclear RNA polymerases of eukaryotic
cells
•There are three distinct classes of RNA pol in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits.
Each class of RNA pol recognizes particular types of genes.

•1. RNA polymerase I: This enzyme synthesizes the precursor of the 28S, 18S, and 5.8S rRNA in the nucleolus.

•2. RNA polymerase II: This enzyme synthesizes the nuclear precursors of mRNA that are subsequently translated to
produce proteins. RNA pol II also synthesizes certain small ncRNAs, such as snoRNA, snRNA and miRNA.

• a. Promoters for RNA polymerase II: In some genes transcribed by RNA pol II, a sequence of nucleotides
(TATAAA) that is nearly identical to that of the Pribnow box is found centered about 25 nucleotides upstream of the
transcription start site. This core promoter consensus sequence is called the TATA, or Hogness, box.

• In the majority of genes, however, no TATA box is present. Instead, different core promoter elements such as Inr
(initiator) or DPE (downstream promoter element) are present (Figure 30.12). [Note: No one consensus sequence
is found in all core promoters.]
• The sequences serve as binding sites for proteins known as general transcription factors (GTFs), which in turn interact
with each other and with RNA pol II.

Figure 30.12 Eukaryotic gene cis-acting promoter

and regulatory elements and their trans-acting
general and specific transcription factors (GTF and
STF, respectively). Inr = initiator; DPE =
downstream promoter element.
•b. General transcription factors
(GTFs): These are the minimal requirements for recognition
of the promoter, recruitment of RNA pol II to the promoter, and
initiation of transcription at a basal level (Figure 30.13A).
•GTFs are encoded by different genes, synthesized in the
cytosol, and transit to their sites of action, and so are
trans-acting.
•[Note: In contrast to the prokaryotic holoenzyme, eukaryotic
RNA pol II does not itself recognize and bind the promoter.
Instead, TFIID, a GTF containing TATA-binding protein and TATA-
associated factors, recognizes and binds the TATA box (and
other core promoter elements). TFIIF, another GTF, brings the
polymerase to the promoter. The helicase activity of TFIIH melts
the DNA, and its kinase activity phosphorylates polymerase,
allowing it to clear the promoter.]

Figure 30.13 A. Association of the general transcription factors (TFIIs) and

RNA polymerase II (RNA pol II) at the core promoters. [Note: The Roman
numeral II denotes the TFs for RNA pol II.] B. Enhancer stimulation of
transcription. CTF = CAAT box transcription factor; Sp1 = specificity factor-1.
•c. Regulatory elements and transcriptional
 Transcriptional activators bind DNA
activators: Upstream of the core promoter are additional through a variety of motifs, such as the
consensus sequences (see Figure 30.12). Those close to helix-loop-helix, zinc finger, and leucine
the core promoter (within 200 nucleotides) are the zipper.

proximal regulatory elements, such as the CAAT and GC

boxes.
•Those farther away are the distal regulatory elements
such as enhancers. Proteins known as transcriptional
activators or specific transcription factors (STFs)
bind these regulatory elements.
•STFs bind to promoter proximal elements to regulate the
frequency of transcription initiation, and to distal elements
to mediate the response to signals such as hormones and
regulate which genes are expressed at a given point in
time. A typical protein-coding eukaryotic gene has binding
sites for many such factors.
•[Note: STFs have two binding domains. One is a DNA-
binding domain, the other is a transcription activation
domain that recruits the GTFs to the core promoter as well
as “coactivator” proteins such as the HAT enzymes
involved in chromatin modification.]
• d. Role of enhancers in eukaryotic gene regulation: Enhancers are
special DNA sequences that increase the rate of initiation of transcription by RNA pol II. Enhancers are
typically on the same chromosome as the gene whose transcription they stimulate
• However, they can:
1) be located upstream (to the 5 -side) or downstream (to the 3 -side) of the transcription start site,
2) be close to or thousands of base pairs away from the promoter (Figure 30.14), and
3) occur on either strand of the DNA.

• Enhancers contain DNA sequences called “response elements” that bind STFs (transcriptional activators).
• By bending or looping the DNA, these enhancer-binding proteins can interact with other transcription
factors bound to a promoter and with RNA pol II, thereby stimulating transcription (see Figure 30.13B).
• [Note: Although silencers are similar to enhancers in that they also can act over long distances, they
reduce gene expression.]

• e. Inhibitors of RNA polymerase II: α-Amanitin, a potent toxin produced by the poisonous
mushroom Amanita phalloides (sometimes called “the death cap”), forms a tight complex with RNA pol II,
thereby inhibiting mRNA synthesis.
•
• 3. RNA polymerase III: This enzyme synthesizes tRNA, 5S rRNA, and some snRNA and snoRNA.
Notes:
• 1- The synthesized RNA will have the sequence of the sense strand
except for U instead of T.
• 2- In prokaryotic (bacteria) all types of RNA are synthesized by
only one species of RNA polymerase.
• 3- In Eukaryotic ( mammaians), there are 3 classes of RNA
polymerase
a- RNA polymerase I: synthesizes the precursor of rRNA named :
pre rRNA
b- RNA polymerase II: synthesizes pre mRNA
c- RNA polymerase III: synthesizes pre tRNA
• All these enzymes synthesize what is called primary transcript or
immature RNAs (pre form) which by some modifications occur
after transcription, will give the mature rRNA, mRNA and tRNA.
Overview of
Transcription :
i. RNA polymerase is the enzyme that controls transcription process
ii. RNA polymerase binds to a promoter region on the DNA
iii. RNA polymerase unwinds the DNA strands & splits it into two strands
iv. RNA polymerase binds free nucleoside triphosphates to the antisense
(template) strand of DNA as it moves along in a 5'≥3' direction
v. using complementary pairing (A with U & C with G) between template
strand and mRNA nucleotides
vi. nucleoside triphosphates loses two phosphates to release the energy
required for transcription process
vii.transcription continues until RNA polymerase reaches a terminator signal
viii.mRNA detaches from the template strand and DNA rewinds
ix. RNA polymerase detaches from the DNA
x. many RNA polymerases can follow each other during transcription process
xi. introns are removed & exons spliced (in eukaryotes) to form mature mRNA
Post-transcriptional modifications of mRNA:
 A primary transcript is the initial, linear, RNA copy of a transcription unit (the segment of DNA between specific initiation
and termination sequences).
 Prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic mRNA is extensively modified both co-
and posttranscriptionally.

Eukaryotic mRNA
The collection of all the primary transcripts synthesized in the nucleus by RNA pol II is known as
heterogeneous nuclear RNA (hnRNA). The pre-mRNA components of hnRNA undergo extensive co- and
posttranscriptional modification in the nucleus. These modifications usually include the following.

a). 5 “Capping”: This is the first of the processing reactions for pre-mRNA. The cap is a 7-methylguanosine attached to
the 5I -terminal end of the mRNA through an unusual 5’ →5’ triphosphate linkage that is resistant to most nucleases.
Creation of the cap requires removal of the g phosphoryl group from the 5 - triphosphate of the pre-mRNA,
followed by addition of guanosine monophosphate (GMP) (from GTP) by the nuclear enzyme
guanylyltransferase. Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-7-
methyltransferase. S-adenosylmethionine is the source of the methyl group.

Additional methylation steps may occur. The addition of this 7-methylguanosine cap helps stabilize the mRNA and permits
efficient initiation of translation.
Role of cap:
a- help to stabilize and Protect mRNA in cytoplasm (how)
b- Permit initiation of translation (specifies, where translation should begin).

b. Addition of a poly-A tail: Most eukaryotic mRNA (with several notable exceptions, including
those coding for the histones) have a chain of 40–250 adenine nucleotides attached to the 3I -end
(see Figure 30.17). This poly-A tail is not transcribed from the DNA, but rather is added after
transcription by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate. The
pre-mRNA is cleaved downstream of a consensus sequence, called the polyadenylation signal
sequence (AAUAAA), found near the 3I -end of the RNA, and the poly-A tail is added to the new 3I -
end.

Role of Poly A-Tail: These tails help stabilize the mRNA, facilitate its exit from the nucleus, and aid
in translation. After the mRNA enters the cytosol, the poly-A tail is gradually shortened.

Posttranscriptional modification of
messenger RNA (mRNA) showing the 7-
methylguanosine cap and poly-A tail.
c. Removal of introns: Maturation of eukaryotic mRNA usually
involves removal from the primary transcript of RNA sequences
(introns, or intervening sequences) that do not code for protein.
The remaining coding (expressed) sequences, the exons, are joined
together to form the mature mRNA. The process of removing introns
and joining exons is called splicing. The molecular complex that
accomplishes these tasks is known as the spliceosome.

1. Role of small nuclear RNAs: In association with multiple proteins,

uracil-rich snRNAs form small nuclear ribonucleoprotein particles
(snRNPs, or “snurps,” designated as U1, U2, U4, U5, and U6) that
mediate splicing. They facilitate the removal of introns by forming base
pairs with the consensus sequences at each end of the intron (Figure
30.18). [Note: In systemic lupus erythematosus, an autoimmune
disease, patients produce antibodies against their own nuclear proteins
such as snRNPs.]

Figure 30.18 Splicing. snRNP = small nuclear ribonucleoprotein particle;

mRNA = messenger RNA. [Note: U1 binds the 5I donor site, U2 binds the
branch A, and addition of U4-U6 completes the complex.]
•Effect of splice site
d. Alternative splicing of mRNA
mutations: Mutations at splice sites can lead
molecules: The pre-mRNA molecules from over 50% of
to improper splicing and the production of
human genes can be spliced in alternative ways in
aberrant proteins. It is estimated that over 15%
different tissues.
of all genetic diseases are a result of mutations
that affect RNA splicing. For example, mutations This produces multiple variations of the mRNA and,
that cause the incorrect splicing of β-globin therefore, of its protein product (Figure 30.19), and thus is
mRNA are responsible for some cases of β- a mechanism for producing a large, diverse set of
thalassemia, a disease in which the production proteins from a limited set of genes.
of the β-globin protein is defective. Splice site
For example, in eukaryotic cells, the mRNA for
mutations can result in exons being skipped
tropomyosin, an actin filament–binding protein of the
(removed) or introns retained. They can also
cytoskeleton (and of the contractile apparatus in muscle
activate cryptic splice sites, which are sites that
cells), undergoes extensive tissue-specific alternative
contain the 5 or 3 consensus sequence but
splicing with production of multiple isoforms of the
aren’t normally used.
tropomyosin protein.

Figure 30.19 Alternative splicing patterns in eukaryotic messenger

RNA.
• There are three major types of RNA that participate in the process of protein synthesis: ribosomal RNA (rRNA),
transfer RNA (tRNA), and messenger RNA (mRNA.
• They are unbranched polymers of nucleotides but differ from DNA by containing ribose instead of deoxyribose
and uracil instead of thymine. rRNA is a component of the ribosomes.
• tRNA serves as an “adaptor” molecule that carries a specific amino acid to the site of protein synthesis.
• mRNA carries genetic information from DNA for use in protein synthesis.
• The process of RNA synthesis is called transcription, and the substrates are ribonucleoside triphosphates.
• The enzyme that synthesizes RNA is RNA polymerase (RNA pol).
• In prokaryotic cells, the core enzyme has five subunits (2α, 1β, 1β , and 1Ω) and possesses 5’ →3’ polymerase
activity that elongates the growing RNA strand.
• This enzyme requires an additional subunit, sigma (s) factor, that recognizes the nucleotide sequence
(promoter region) at the beginning of a length of DNA that is to be transcribed. This region contains consensus
sequences that are highly conserved and include the TATA (Pribnow) box and the –35 sequence.
• Another protein—rho (r) factor—is required for termination of transcription of some genes.
• There are three distinct classes of RNA pol in the nucleus of eukaryotic cells. RNA pol I synthesizes the
precursor of rRNAs in the nucleolus. In the nucleoplasm, RNA pol II synthesizes the precursors for mRNA and
some noncoding RNAs, and RNA pol III produces the precursors of tRNA.
• In both prokaryotes and eukaryotes, RNA pol does not require a primer and has no 3I →5I exonuclease
(proofreading) activity.
• Core promoters for genes transcribed by RNA pol II contain cis-acting consensus sequences, such as
the TATA-like Hogness box, that serve as binding sites for trans-acting general transcription factors.
• Upstream of these are proximal regulatory elements, such as the CAAT and GC boxes, and distal
regulatory elements such as enhancers.
• Transcriptional activators (specific transcription factors) bind these elements and regulate the
frequency of transcription initiation, the response to signals such as hormones, and which genes are
expressed at a given point in time.
• Eukaryotic transcription requires that the chromatin be accessible.
• A primary transcript is a linear copy of a transcription unit, the segment of DNA between
specific initiation and termination sequences.
• The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally
modified by cleavage of the original transcripts by ribonucleases.
• rRNAs of both prokaryotic and eukaryotic cells are synthesized from long precursor molecules called
pre-rRNA. These precursors are cleaved and trimmed by ribonucleases, producing the three largest
rRNA, and bases and sugars are modified.
• Eukaryotic 5S rRNA is synthesized by RNA pol III and is modified separately.
• Prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic mRNA is
extensively modified co- and posttranscriptionally.
• For example, a 7-methylguanosine cap is attached to the 5I -terminal end of
the mRNA through a 5’ →5’ linkage.
• A long poly-A tail, not transcribed from the DNA, is attached to the 3’ -end of
most mRNAs.
• Most eukaryotic mRNAs also contain intervening sequences (introns) that
must be removed to make the mRNA functional.
• Their removal, as well as the joining of expressed sequences (exons),
requires a spliceosome composed of small, nuclear ribonucleoprotein
particles (snurps) that mediate the process of splicing.
• Eukaryotic mRNA is monocistronic, containing information from just one
gene.
• Prokaryotic and eukaryotic tRNAs are also made from longer precursor
molecules.
• If present, an intron is removed by nucleases, and both ends of the molecule
are trimmed by ribonucleases.
• A 3I -CCA sequence is added, and bases at specific positions are modified,
producing “unusual” bases.
Key concept map for RNA structure and
synthesis. rRNA = ribosomal RNA; tRNA =
transfer RNA; mRNA = messenger RNA.
By: Dr. Arika,
Ph.D