RNA Synthesis and Processing

• The process by which RNA is synthesized from DNA template is called

transcription.
• Of the two strands of DNA, one acts as template strand (antisense
strand) and the other is nontemplate strand (or coding strand or sense
strand)
• RNA is synthesized from its 5′-end to its 3′-end, antiparallel to its DNA
template strand (3’-end to 5’-end). The template is copied as
complementary bases to form new RNA strand, in which a G on the DNA
specifies a C in the RNA, a C specifies a G, a T specifies an A, but an A
specifies a U instead of a T.

• The following is an example:

• The process is catalyzed by enzyme RNA Polymerase. RNA

polymerase does not require a primer, and has no proofreading
activity.
A. I n Prokaryotes - RNA Polymerase- The enzyme that synthesizes
all types of RNAs. 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 (σ) factor—that recognizes the nucleotide sequence (promoter
region) at the beginning of a length of DNA that is to be transcribed.

i. Core enzyme: Four RNA polymerase enzyme subunits, 2α, 1β,

and 1β', which are responsible for the 5′→3′ RNA polymerase
 activity, and are called to core enzyme. They do not recognize
promoter region on DNA.
ii. Holoenzyme: The σ subunit (“sigma factor”) recognizes
promoter regions on the DNA. The σ subunit plus the core
enzyme make up the holoenzyme.

○ The Promotor region is in beginning of the DNA template contains

characteristic consensus nucleotide sequences that are highly
conserved and include the Pribnow box and the –35 sequence.
a. Pribnow box: This is a stretch of six nucleotides (5′-TATAAT-3′)
centered about ten nucleotides to the left of the transcription
start site. [.The first base at the transcription start site is
assigned a position of +1. The Pribnow box is centered at
approximately base –10. There is no base designated “0.”]
b. –35 sequence: A second consensus sequence (5′-TTGACA-3′), is
centered about 35 bases to the left of the transcription start site.
See in fig below promoter region of DNA template

• Another protein—rho (ρ) factor—is required for termination of

transcription of some genes.

B. In Eukaryotes- There are three distinct classes of RNA

polymerase in the nucleus of eukaryotic cells.

(i) RNA polymerase I synthesizes the precursor of large rRNA in the

nucleolus(the 28S, 18S, and 5.8S r-RNA)

(ii) RNA polymerase II synthesizes the precursors for mRNA, and

(iii) RNA polymerase III produces the precursors of tRNA. .

• Promoters for genes transcribed by RNA polymerase II contain

consensus sequences, such as the TATA or Hogness box, the CAAT
box, and the GC box. They serve as binding sites for proteins called
general transcription factors, which, in turn, interact with each other
and with RNA polymerase II.
• Enhancers are DNA sequences that increase the rate of initiation of
transcription by binding to specific transcription factors called activators
• A transcription unit extends from the promoter to the termination
region, and the product of the process of transcription by RNA
polymerase is termed the primary transcription.
• The promoter sequences recognized by RNA polymerase II (in
eukaryotes) are-
(i) TATA or Hogness box –It is a promoter sequence of
nucleotides (similar to Pribnow box) and is found centered
about 25 nucleotides upstream of the transcription start site.
(ii) A second consensus sequence known as the CAAT box is
found between 70 and 80 nucleotides upstream of the
transcription start site.
(iii) In other genes, , a GC-rich region (GC box) is found.
Such sequences serve as binding sites for proteins known as
transcription factors, which in turn interact with each other and with
RNA polymerase II. See in fig below- Eukaryotic gene promoter
consensus sequences.

○ Transcription factors - Transcription factors recognize the

promoter, pull RNA polymerase II to the promoter, and initiate
transcription in eukaryotes.. They are CTF, SP I, TFIID, Pol lI, They
bind with promoter (TATA, CAT and GC sites) to form transcription
initiation complex

○ Enhancers- May be present far from the promoter. By bending of

DNA the far placed Enhancers interacts with the transcription
initiation complex. See Fig next page
 ○

C. Steps if Transcription-
1. Initiation- Transcription starts by the binding of RNA polymerase
holoenzyme to the promoter region of the DNA.

2. Elongation-
• When the promoter region is
bound by the holoenzyme, local
unwinding (melting) of the DNA
helix occurs. Positive and negative supercoils are formed in DNA
which are relieved by tropisomerases I & II. see fig next page
• RNA polymerase begins to synthesize several short pieces of RNA
transcripts of the DNA sequence, and are initially discarded.
• The elongation phase is said to begin when the transcript (typically
starting with a purine) exceeds 10 nucleotides in length.
• The core RNA polymerase enzyme leave the promoter and move
along the DNA template strand in a processive manner.
• During replication, a short DNA-RNA hybrid helix is formed.
• The transcription is always in the 5′→3′ direction.
See in Fig Below- Local unwinding of DNA caused by RNA polymerase.-
see template & nontemplate strands of DNA, ;positive and negative
supercoils of DNA formed on unwinding,, RNA-DNA hybrid helix,
position of RNA polymerase enzyme, new RNA being formed
complementary to template DNA strand. Elongation at 3’ end of new
RNA-

Make this figure (M Impt)

3. Termination: The elongation of the single-stranded RNA chain

continues until a termination signal is reached. Termination can be
pontaneous or dependent upon the participation of a protein known as
the ρ (rho) factor.

a. ρ-Independent termination: The newly formed RNA fold back on itself,

forming a GC-rich stem (stabilized by H-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 3′-end. The bonding
of these Us to the complementary strand of the DNA template is weak.
 This facilitates the separation of the newly synthesized RNA from its
DNA template.

b. ρ-Dependent termination: This requires the participation of an

additional protein, rho = ρ factor, which has ATPase with helicase
activity. It binds a C-rich “rho recognition site” near the 3′-end of the
newly formed RNA and, using its ATPase activity, moves along the RNA
until it reaches the RNA polymerase paused at the termination site. The
ATP-dependent RNA-DNA helicase activity of rho separates the RNA-
DNA hybrid helix, causing the release of the RNA.

Post-trancriptional Modifications-
• The primary transcripts of both prokaryotic and eukaryotic tRNA and
rRNA are posttranscriptionally modified by cleavage of the original
transcripts by ribonucleases. rRNA of both prokaryotic and eukaryotic
cells are synthesized from long precursor molecules called
preribosomal RNA. These precursors are cleaved and trimmed by
ribonucleases, producing the three largest rRNA. (Eukaryotic 5S rRNA
is synthesized by RNA polymerase III instead of I, and is modified
separately.)
• Prokaryotic mRNA is generally identical to its primary transcript,
whereas eukaryotic mRNA is extensively modified posttranscriptionally.
For example, a 7-methylguanosine “cap” is attached to the 5′-terminal
end of the mRNA through a triphosphate linkage, resulting in a 5′→5′
linkage. A long poly-A tail—not transcribed from the DNA—is attached
to the 3′-end of most mRNA. Many 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 small, nuclear ribonucleoprotein
particles that mediate the process of splicing.
• Prokaryotic and eukaryotic tRNA are also made from longer precursor
molecules. These must have an intron removed, and the 5′- and 3′-ends
of the molecule are trimmed by ribonuclease. A 3′-CCA sequence is
added, and bases at specific positions are modified, producing
“unusual” bases.

Posttranscriptional Modification of RNA

• The primary transcripts of both prokaryotic and eukaryotic tRNA and
rRNA are posttranscriptionally modified by ribonucleases
(RNases)..
• Prokaryotic mRNA is generally identical to its primary transcript ,
whereas eukaryotic mRNA is extensively modified
posttranscriptionally.
A. Ribosomal RNA
• r-RNA is synthesized from long precursor molecules called pre -
ribosomal RNAs. The pre-ribosomal RNA are cleaved by ribonucleases
to yield intermediate-sized pieces of rRNA, which are further “trimmed”
to produce the required RNA species. The 28S, 18S, and 5.8S rRNA
are produced in eukaryotes (See Fig Below).
Posttranscriptional processing of eukaryotic ribosomal RNA by
ribonucleases (RNases).

B. Transfer RNA
Both eukaryotic and prokaryotic tRNAs are also made from longer
precursor molecules that must be modified as under-. .
a. An intron must be removed from the anticodon loop,
b. sequences at both the 5′- and the 3′-ends of the molecule must be
trimmed and
c. a –CCA sequence is added by nucleotidyl-transferase at the 3′-
terminal end of tRNA,
See in fig - A. Primary tRNA transcript. B. Functional tRNA after
posttranscriptional modification. Modified bases include D
(dihydrouracil), ψ (pseudouracil), and , which means that the base
m

has been methylated.

C. Eukaryotic mRNA
The primary script of mRNA molecule synthesized by RNA polymerase II is
known as heterogeneous nuclear RNA (hnRNA). The primary transcripts
are extensively modified in the nucleus after transcription. These
modifications usually include:

a. 5′ “Capping”: This process is the first of the processing reactions

for hnRNA (Figure next page). The cap is a 7-methylguanosine
attached “backward” to the 5′-terminal end of the mRNA. The
creation of the guanosine triphosphate part of the cap requires the
nuclear enzyme guanyly-ltransferase. Methylation of this terminal
guanine is catalyzed by guanine-7-methyltransferase. The addition
of this 7-methylguanosine “cap” permits the initiation of translation,
and helps stabilize the mRNA. Eukaryotic mRNA lacking the cap are
not efficiently translated.

Posttranscriptional modification of mRNA showing the 7-

methylguanosine cap and poly-A tail.
 b. Addition of a poly-A tail: Most eukaryotic mRNA have a chain of
40–200 adenine nucleotides attached to the 3′-end .. This poly-A tail
is added after transcription by the nuclear enzyme,
polyadenylate polymerase, using ATP as the substrate. The mRNA
is cleaved downstream of a consensus sequence, called the
polyadenylation signal sequence (AAUAAA), found near the 3′-end
of the RNA, and the poly-A tail is added to the new 3′-end. These
tails help stabilize the mRNA and facilitate their exit from the
nucleus. After the mRNA enters the cytosol, the poly-A tail is
gradually shortened.
c. Removal of introns: Maturation of eukaryotic mRNA usually involves
the removal of RNA sequences, which do not code for protein
(introns, or intervening sequences) from the primary transcript.
The remaining coding sequences, the exons, are joined together
to form the mature mRNA. The process of removing introns and
joining exons is called splicing. The molecular machine that
accomplishes these tasks is known as the spliceosome.

○ Role of snRNAs: small nuclear ribonucleoprotein particles (snRNP, or

“snurps”) mediate splicing. They facilitate the removal of exon
segments by forming base pairs with the consensus sequences at
each end of the intron

○ Mechanism of splicing: The binding of snRNP brings the sequences

of the neighboring exons into the correct alignment for splicing. The
2′-OH group of an adenosine (A) residue (known as the branch site) in
the intron attacks the phosphate at the 5′-end of the intron, forming an
 unusual 2′→5′ phosphodiester bond and creating a “lariat” structure
(see Figure 30.18). The newly freed 3′-OH of exon 1 attacks the 5′-
phosphate at the splice acceptor site, forming a phosphodiester bond
that joins exons 1 and 2. The excised intron is released as a lariat,
which is degraded. After introns have been removed and exons joined,
the mature mRNA molecules leave the nucleus and pass into the
cytosol through pores in the nuclear membrane.

• Effect of splice site mutations: Mutations at splice sites can lead to

improper splicing and the production of aberrant proteins. For
example, mutations that cause the incorrect splicing of β-globin
mRNA are responsible for som cases of β-thalassemia—a disease in
which the production of the β-globin protein is defective.

• Systemic lupus erythematosus, a fatal inflammatory disease, results

from an autoimmune response in which the patient produces antibodies
against host proteins, including snRNP.

○ See next page figure- Splicing. snRNP = small nuclear

ribonucleoprotein particle.
Study Questions
Choose the ONE correct answer.
30.1 A one-year-old male with chronic anemia is found to have β-
thalassemia. Genetic analysis shows that one of his β-globin genes
has a G to A mutation that creates a new splice acceptor site nineteen
nucleotides upstream from the normal splice acceptor site of the first
intron. Which of the following best describes the new messenger RNA
molecule that can be produced from this mutant gene?
A. Exon 1 will be too short.
B. Exon 1 will be too long.
C. Exon 2 will be too short.
D. Exon 2 will be too long.
E. Exon 2 will be missing.
Hide Answer
Correct answer = D. Because the mutation adds an additional
splice acceptor site (the 3′-end) of intron 1 upstream, the
nineteen nucleotides that are usually found at the 3′-end of the
excised intron 1 lariat can remain behind as part of exon 2 as a
result of aberrant splicing. Exon 2 can, therefore, have these
extra nineteen nucleotides at its 5′-end. The presence of these
extra nucleotides in the coding region of the mutant m-RNA
molecule will prevent the ribosome from translating the message
into a normal β-globin protein molecule. Those mRNA for which
the normal splice site is used to remove the first intron will be
normal, and their translation will produce normal β-globin
protein.

30.2 The base sequence of the strand of DNA used as the template for
transcription has the base sequence GATCTAC. What is the base
sequence of the RNA product? (All sequences are written according to
standard convention.)
A. CTAGATG.
B. GTAGATC.
C. GAUCUAC.
D. CUAGAUG.
E. GUAGAUC.
Hide Answer
Correct answer = E. All sequences are written in the standard
convention (5′→3′). The RNA product has a sequence that is
complementary to the sequence of the template strand of DNA.
Uracil (U) is found in RNA in place of the thymine (T) in DNA.
Thus, the DNA template 5′-GATCTAC-3′ would produce the RNA
product 3′-CUAGAUG-5′ or, written correctly in the standard
direction, 5′-GUAGAUC-3′.

30.3 A four-year-old child who becomes easily tired and has trouble
walking is diagnosed with Duchenne muscular dystrophy, an X-linked
recessive disorder. Genetic analysis shows that the patient's gene for
the muscle protein dystrophin contains a mutation in its promoter
region. Of the choices listed, what would be the most likely effect of
this mutation?
A. Initiation of dystrophin transcription will be defective.
B. Termination of dystrophin transcription will be defective.
C. Capping of dystrophin mRNA will be defective.
D. Splicing of dystrophin mRNA will be defective.
E. Tailing of dystrophin mRNA will be defective.
Hide Answer
Correct answer = A. Mutations in the promoter prevent formation
of the RNA polymerase II transcription complex, and the
initiation of mRNA synthesis will be greatly decreased. A
deficiency of dystrophin mRNA will result in a deficiency in the
production of the dystrophin protein.

30.4 A mutation to this sequence in eukaryotic mRNA will affect the

process by which the 3′-end poly-A tail is added to the mRNA.
A. CAAT
B. CCA
C. GGGGCG
D. AAUAAA
E. TATAAA
Hide Answer
Correct answer = D. An endonuclease cleaves mRNA just
downstream of this polyadenylation signal, creating a new 3′-end
to which the pol A polymerase adds the poly-A tail using ATP as
the substrate in a template-independent process. CAAT,
GGGGCGT, and TATAAA are sequences found in promoters for
RNA polymerase II. CCA is added to the 3′-end of tRNA by
nucleotidyl transferase. 7-CH 3 guanosine is part of the cap
structure at the 5′-end of eukaryotic mRNA.