RNA TRANSCRIPTION

Transcription is the synthesis of an RNA molecule from a DNA template strand.

Transcription occurs in the nucleoid region of the cytoplasm in bacterias.
Three Steps of RNA Transcription:

1. Initiation
2. Elongation
3. Termination

INITIATION
What occurs during initiation?

1. Transcription enzyme, called RNA polymerase, along with the general transcription factors
assemble on the promoter DNA to form a transcription pre-initiation complex (PIC).
2. The TATA box, as a core promoter element, is the binding site for a transcription factor known as
TATA-binding protein (TBP). TFIID binds to the TATA box via the TBP, which itself is a subunit of
TFIID.
3. Five more transcription factors and RNA polymerase combine around the TATA box in a series of
stages to form a pre-initiation complex.
4. Two transcription factors, Transcription Factor II H (TFIIH) and TFIIE, are involved in separating
opposing strands of double-stranded DNA to provide the RNA polymerase access to a single-
stranded DNA template.

Initiation of transcription differs from initiation of DNA replication in several ways. One difference is that
initiation of transcription does not require a primer.

How did TATA box get its name?

It was termed the "TATA box" as it contains a consensus sequence characterized by repeating T and A
base pairs.

How does DNA molecule unwound in transcription?

Previous studies found that the transcription factors TFIIE and TFIIH are required for this transition. Dr.
Sebastian Grünberg and colleagues in the Hahn Lab of the Basic Sciences Division found that the Tfa1
and Tfa2 subunits of TFIIE heterodimerize through their winged helix motifs, forming a unique triple
winged helix fold. When incorporated into a working structural model of the pre-initiation complex, this
fold spans the Pol II active site cleft, thereby encircling a region of ds-DNA that is upstream of the
transcription start site. Ssl2 in the pre-initiation complex translocates the template strand (and
transcription start site) into the Pol II cleft by a right-handed threading mechanism, contrary to just
 rotating the DNA. Threading of the DNA displaces the non-template strand onto the DNA-binding
domain(s) of TFIIE and generates an open complex.

Reference: https://www.fredhutch.org/en/news/spotlight/imports/how-dna-unwinds-at-the-onset-of-
transcription.html

Within the transcription bubble, the 9 most recently added nucleotides in the newly synthesized RNA strand
temporarily form a helix with the template DNA strand. How might transcription be affected if helix formation
did not occur?

The position of the 3' end of the RNA would be unstable, inhibiting elongation.

ELONGATION
What occurs during elongation?

1. Elongation involves synthesis of mRNA in the 5′ to 3′ direction at a rate of approximately 40

nucleotides per second.
2. RNA polymerase traverses the template strand from 3’ to 5’ direction.
3. RNA polymerase uses base pairing complementarity with the DNA template.
4. An RNA molecule from 5' → 3' direction is produced. It is an exact copy of the coding strand
(except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-
carbon) sugar where DNA has deoxyribose (one fewer oxygen atom) in its sugar-phosphate
backbone).
5. Unlike DNA replication the DNA molecule re-winds by itself (hydrogen bonds bind again) to form its
double helix.

Thymine is the nitrogenous base found in DNA but not in RNA.

Uracil is the substance found in RNA but not in DNA.

Why does the RNA polymerase write mRNA from the 5’ to 3’ direction?

RNA polymerase can only add nucleotides to the 3' end of the strand so like DNA, RNA must be
synthesized in the 5' to 3' direction.

Why is thymine replaced by uracil in RNA synthesis?

It's important for the cell to distinguish message (RNA) from gene (DNA) in order to preserve the genes
intact in an environment where the message is continually broken down and resynthesized.
Substituting U for T provides another criterion of specificity to ensure that RNAases will not attack the
DNA sequence.
 TERMINATION
What occurs during termination?

1. RNA polymerase continues to elongate until it reaches the terminator, a specific sequence of
nucleotides that signals the end of transcription.
2. The transcript is cleaved at an internal site releasing the upstream portion of the transcript, which
will serve as the initial RNA prior to further processing (pre-mRNA).
3. The termination sequence usually consists of a series of adjacent adenines preceded by a
nucleotide palindrome giving an RNA molecule that assumes a stem-and loop configuration.
This configuration stops RNA polymerase from transcribing any further.

Termination begins when a polyadenylation signal appears in the RNA transcript.

A polyadenylation signal is a sequence of nucleotides that mark where the RNA transcript should end; it
is positioned after the stop codon.

A stop codon is a nucleotide triplet within messenger RNA. When these codons undergo RNA
transcription, they change to UAA (uracil - adenine - adenine), UAG (uracil - adenine - guanine), and UGA
(uracil - guanine - adenine).

Since the pre-mRNA formed is still bound to the RNA polymerase II, it needs to be cleaved in order to
complete the maturing of the pre-mRNA.

The cleavage site is a small distance from the termination signal.

Cleavage and polyadenylation specificity factor (CPSF) is a protein complex responsible for the
cleavage of the 3’ signaling region in our mRNA.

What 3 events occur in eukaryotic RNA processing?

1. CAPPING - guanine nucleotide is added to the end of RNA molecule

2. POLYADENYLATION - when RNA polymerase reaches end of gene, termination proteins add on
about 200 adenine nucleotides to the end of the RNA molecule
3. SPLICING - newly formed, "pre-messenger RNA" contains INTRONS, which are removed by
spliceosomes to produce a functional RNA molecule (containing only coding regions, or exons),
which is ready to leave the nucleus

After the cleavage occurs, an enzyme called polynucleotide adenyltransferase or polyadenylate

polymerase (PAP) will be responsible for the addition of 3’ poly-A tail to the pre-mRNA. The final tail is
about 200-250 adenine nucleotides long.

The rate at which PAP adds adenine nucleotides is dependent on the presence of polyadenylate binding
protein II (PABPII), which is a regulatory protein.
5’ cap is a modified guanine (G) nucleotide, called 7-methylguanosine. It protects the transcript from being
broken down. It also helps the ribosome attach to the mRNA and start reading it to make a protein.

Importance of 5’ cap and poly-A tail:

1. prevents the degradation of pre-mRNA

2. promotes nuclear transport of pre-mRNA from nucleus to cytoplasm
3. promotes translation

RNA splicing is the editing of the nascent pre-mRNA transcript into a mature messenger RNA.

Steps in RNA splicing:

1. As soon as a pre-mRNA is transcribed, it is quickly bound by several complexes, called snRNPs.

2. snRNPs bind to sites in a pre-mRNA at or near the intron-exon boundaries. These sites, called
consensus sequences, contain nucleotide sequences that are shared by most pre-mRNAs. The
snRNPs contain RNA molecules that can bind to these consensus sequences through
complementary base pairing.
3. In addition to the snRNPs that have bound to the consensus sequences, other snRNPs attach to
other sequences within the intron. The snRNPs eventually come together into a large complex
called a spliceosome. As the spliceosome forms, the intron loops out.
4. The spliceosome cuts the pre-mRNA at one intron-exon boundary, where it leaves a reactive free
hydroxyl (–OH) group on the exon. The spliceosome uses this hydroxyl group to attack the other
end of the intron and in the process removes of the intron and joins the exons together, forming a
mature mRNA molecule.
5. The mRNA leaves the nucleus and is translated into protein within the cytoplasm. The intron that
remains is quickly degraded. The snRNPs remain in the cell and are used to splice introns from
other RNA molecules.

During the splicing reaction, the intron-exon junctions are recognized by small nuclear
ribonucleoproteins or snRNPs, which are subunits of spliceosome.

How are introns recognized?

Introns contain marker sequences at both of their ends(GU nucleotides at the 5’ end and AG
nucleotides at the 3’ end). These are recognized by the snRNPs and direct the spliceosome to remove
the intron.

Spliceosome is an enzyme complex made of protein and small RNAs. It removes intronic sequences from
primary transcripts to generate functional messenger and long noncoding RNAs.

The portion of the DNA molecule that is not translated and is a noncoding portion of DNA is composed of
introns while the portion of the DNA molecule that is translated is composed of exons.

Why should introns be removed from the mRNA?

Introns have to be removed in order for the mRNA to encode a protein with the right sequence. If the
spliceosome fails to remove an intron, an mRNA with noncoding portions in it will be made, and a
wrong protein is will get produced during translation.

Prokaryotic vs. Eukaryotic Transcription

 LOCATION: transcription occurs in  LOCATION: transcription occurs in the

cytosol
 INITIATION: contain three different
promoter elements: -10, -35 promoters,
and upstream elements
 only one type of RNA polymerase
 no pre-initiation complex
 transcription and translation occur
simultaneously
 TERMINATION: done by either rho-
dependent or rho-independent
mechanisms
nucleus
 INITIATION: contain many different
promoter elements
 have three types of RNA polymerases, I,
II, and III
 forms pre-initiation complex
 RNA is first transcribed in the nucleus
and then translated in the cytoplasm
 TERMINATION: terminated by two
elements: a poly(A) signal and a
downstream terminator sequence