TRANSCRIPTION

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RNA polymerase carries out transcription. Transcription is the process of linking ribonucleotide to

form long chain or to form oligonucleotide. The process of synthesizing

RNA from DNA molecule.

The 2’-OH of the ribose sugar make phosphodiester bond unstable and

that causes ssRNA easy to be degraded. Cell has mechanism to remove

mRNA that is not correct.

Transcription VS Replication

- Transcription initiates both of prokaryotic and eukaryotic cell

- Form more sites than replication

- The number of RNAP molecules are more than number of

DNAP molecules

- RNAP proceed at slower rate than DNAP (~ 50-100 bases/sec for RNAP, ~ 1000 bases/sec for

DNAP)

- The fidelity of RNAP lower than DNAP

Allowed since the aberrant RNA molecules can simply be turned over and new correct

molecules can be made. (true for Prok. But not Euk.)

**Replication happens too fast and cause lots of error but DNAP has exonuclease activity to correct

the error.

Transcription has three steps (Initiation, Elongation and Termination)

Before proceeds to the steps, let us look at the Promoter.

Promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located

near the genes they transcribe, on the same strand and upstream on the DNA (towards the 3' region

of the anti-sense strand, also called template strand and non-coding strand). Promoters can be about

100–1000 base pairs long.

Promoters contain specific DNA sequences and response elements that provide a secure initial

binding site for RNA polymerase and for proteins called transcription factors that recruit RNA

polymerase.

In bacteria - The promoter is recognized by RNA polymerase and an associated sigma factor, which in

turn are often brought to the promoter DNA by an activator protein's binding to its own DNA binding

site nearby.

In eukaryotes - The process is more complicated, and at least seven different factors are necessary

for the binding of an RNA polymerase II to the promoter.

TRANSCRIPTION

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In bacteria, the promoter contains two short sequence elements approximately -10 and -35

nucleotides upstream from the transcription start site.

The sequence at -10 (the -10 element) has the consensus sequence TATAAT.

The sequence at -35 (the -35 element) has the consensus sequence TTGACA.

The nucleotide at which transcription is initiated is denoted as +1. The preceding nucleotide as -1.

Those portions of the DNA preceding the initiation site (toward the 3’ end of the template) are said

to be upstreamfrom that site. Those portions of the DNA succeeding it (toward the 5’ end of the

template) are said to be downstream from that site.

The RNAP is a holoenzyme that consist of 5 subunits

Rifampicin – a semi synthetic antibiotic derived from the original molecule (rifamycin – isolated from

streptomycete). Binds to the beta (β) subunit of RNAP and inhibits the formation of first

phosphodiester bond. Act on prok. and euk.

The sigma (σ) factor

- Proteins that are required for the binding of the RNA polymerase to the promoter so the

transcription can be initiated.

- Recognizes promoter and facilitates initiation.

- Facilitate the binding of RNAP to dsDNA.

- To direct RNAP to transcribe DNA template at the correct transcription start site (TSS).

Function of sigma (σ) factor

- Ensure RNAP binds stably to DNA only at promoter (not string bind) at first but then if reach

prmoter sequence the strong binding)

- Destabilize nonspecific binding to nonpromoter DNA

- Stabilize specific binding to promoter DNA

- Accelerates the search for promoter DNA

TRANSCRIPTION

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RNA polymerases are not capable of recognizing promoters on their own but require the help of

additional proteins called transcription factors

(in Eukaryotes)

The polymerase moves along the template DNA

strand towards its 5’ end (3’ 5’ direction).

Therefore, the resultant pre-mRNA transcript

grows from the 5’ end. The DNA is temporarily

unwound as the polymerase progresses and

reform the double helix after the polymerase

has moved past a particular stretch of DNA.

Chain elongation occurs as a result of an attack by

3’ OH of the nucleotide at the end of the growing

strand on the 5’ α-phosphate of the incoming

nucleoside triphospahte

For each gene, only one of the two DNA strand is

transcribed. This strand is called the template

strand. Note that for every particular gene, the

same strand is used as the template every time it

is transcribed. The mRNA base triplets are called

codons. the nontemplate strand is sometimes

called the coding strand. The nontemplate

sequences are same as the sequence of mRNA

except for the T is replaced by U.

TRANSCRIPTION

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Transcription in Prokaryotes

As what has been mentioned before, bacteria such

as E.coli contain RNA polymerase composed of 5

subunits. Without the σ subunit, it is called as core

enzyme. Before the enzyme (RNAP) attaches itself to

the DNA, σ factor is added. Attachment of σ factor

to the core enzyme increases the enzyme’s affinity

for promoter sites in DNA and decreases its affinity

for DNA in general. Once bound to the promoter,

the enzyme separates (melt) the two DNA strands in

the region surrounding the start site, which make

the template strand accessible to the enzyme’s

active site. One approximately ten nucleotides have

been successfully incorporated into a growing

transcript; the enzyme undergoes a conformation

change which lead to the release of σ sigma factor

and the promoter DNA. This will make the enzyme

more stable and is converted into an elongation

complex.

Bacterial cells possess a variety of different σ factors that

Recognize different versions of the promoter sequence. The σ70 is known as the “housekeeping” σ

factor, because it initiates transcription of most genes. Alternative σ factors initiate transcription of a

small number of specific genes that participate in a common response. For example, when E. coli

cells are subjected to a sudden rise in temperature, a new σ factor is synthesized that recognizes a

different promoter sequence and leads to the coordinated transcription of a battery of heat-shock

genes.

To terminate the transcription, a specific nucleotide sequence is needed. Terminations involve two

cases, which are rho-dependent termination and rho-independent termination (also known as

intrinsic termination).

Rho (ρ)-independent termination

These termination sequences have two characteristics

features:

- A series of U residues in the transcribed RNA

- GC rich self-complementary region with several

intervening nucleotides

When the self-complementary region of a growing RNA

chain is synthesized, the complementary sequences

base-pair with one another, forming a stem-loop structure (hairpin loop).

TRANSCRIPTION

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This stem-loop structure is thought to interact with the transcribing E.coli RNA polymerase, causing it

to pause. The base pair between the U residues at the 3’ end of the nascent RNA chain and the A

residues in the template DNA strand are extremely unstable compared to other types of Watson-

Crick base pairs. If the base pairs is replaced or substituted with other bases, the terminator function

may decreases because the RNA-DNA hybrid duplex will be more stable. These two features probably

permit release of the RNA chain from the transcription complex at Rho-independent termination

sites.

Rho (ρ)-dependent termination

- Rho factor is a hexameric protein which wraps around the 70-to 80-base segment of the

growing RNA transcript

- Activates the ATPase activity of Rho that is associated with its movement along the RNA in

the 3’ direction until it eventually unwinds the RNA-DNA hybrid (break H-bond) at the active

site of RNAP.

- Pausing of polymerase during elongation is thought to be an important component of Rho-

dependent termination as it is in Rho-independent termination.