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. In 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.

o 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 init iation 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.

(i i) A second consensus sequence known as the CAAT box is

found between 70 and 80 nucleotides upstream of the

transcription start site.

(i i i) 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.

o Transcription factors - Transcription factors recognize the

promoter, pull RNA polymerase II to the promoter, and init iate

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

o 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

o

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 (stabil ized 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 facil itates 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′ l inkage. 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 m, which means that the base 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 init iation of translation,

and helps stabil ize 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.

o 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

o 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.

o 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. Init iation 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. Tail ing 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.