Transcription in prokaryotes

E. coli RNA polymerase

• It consists of four kinds of subunits.
• The subunit composition of the enntire
enzyme, called holoenzyme, is α2ββ’σ.
• The σ subunit finds a promoter site where
transcription begins, helps initiate RNA
synthesis and then dissociates from the rest of
the enzyme.
• The enzyme without this subunit is called core
enzyme.
 E. coli RNA polymerase
• The σ subunits bind regulatory proteins.
• The β’ subunit binds the DNA template.
• The β subunit binds ribonucleotides
substrates.
 E. coli RNA polymerase
• The synthesis of RNA by E. coli RNA
polymerase takes place in three stages:
initiation, elongation, and termination.
• It performs multiple functions in this process:
1. It searches DNA for initiation sites.
2. It unwinds a short stretch of double helical
DNA to produce a single stranded DNA
template from which it takes instructions.
 E. coli RNA polymerase
• It selects the correct ribonucleotide and catalyzes
the formation of a phosphodiester bond. This
process is repeated many times as the enzyme
moves unidirectionally along the DNA template.
RNA polymerase is totally processive – a transcript
is synthesized from start to end by a single RNA
polymerase molecule.
• It detects termination signals that specify where a
transcript ends.
• It interacts with activator and repressor proteins
that modulate the rate of transcription over a wide
dynamic range.
Transcription is initiated at promoter
sites on the DNA template
• Transcription starts at promoters on the DNA
template.
• Two common motifs are present on the 5’
(upstream) side of the start site. They are
known as the -10 sequence and the -35
sequence.
Transcription is initiated at promoter
sites on the DNA template
• The sequence of the template strand of DNA is
the complement of that of the RNA transcript.
• The coding strand of DNA has the same
sequence as that of the RNA transcript except
for T in place of U.
• The coding strand is also known as the sense
(+) strand and the template strand as the
antisense (-) strand.
RNA polymerase unwinds nearly two
turns of template DNA before
intiating RNA synthesis
• Each pound polymerase unwinds a 17-bp
segment of DNA, which corresponds to 1.6
turns of B-DNA helix.
• Most newly synthesized RNA chains carry a
tag that reveals how RNA chains are started.
• The 5’ end of a new RNA chain is highly
distinctive: the molecule starts with either
pppG or pppA.
• In contrast with DNA synthesis, a primer is not
needed.
• RNA chains can be formed de novo.
• RNA chains grow in the 5’→3’ direction.
• The elongation phase of RNA synthesis begins
after formation of the first phosphodiester
bond.
• An important change is the loss of σ –the core
enzyme without σ binds more strongly to the
DNA template.
• RNA polymerase stays bound to its template
until a termination signal is reached.
• The region containing RNA polymerase, DNA,
and the nascent RNA is called a transcription
bubble because it contains a locally melted
bubble of DNA.
It is noteworthy that RNA polymerase lacks nuclease activity. In
contrast with DNA polymerase, it does not edit the nascent
polynucleotide chain.
Consequently, the fidelity of transcription is much lower than that
of replication.
• In the termination phase of transcription, the
formation of phosphodiester bonds ceases,
the RNA-DNA hybrid dissociates, the melted
region of DNA rewinds, and RNA plymerase
releases the DNA.
• The transcribed regions of DNA templates
contain stop signals.
• The simplest one is a palindromic GC-rich
region followed by an AT rich region.
• The RNA transcript of this DNA palindrome is
self complementary.
 Types of RNA in prokaryotes
• In prokaryotes, mRNA molecules undergo
little or no modification following synthesis by
RNA polymerase.
• Many of them are translated while they are
being transcribed.
• In contrast, transfer RNA and ribosomal RNA
molecules are generated by cleavage and
other modifications of nascent RNA chains.
 Types of RNA in prokaryotes
• In E. coli, three kinds of ribosomal RNA
molecules and a transfer RNA molecule are
excised from a single primary RNA transcript
that also contains spacer region (yellow).
 Types of RNA in prokaryotes
• CCA is added to the 3’ end of tRNA molecules
that do not already posses this terminal
sequence.
• Unusual bases are found in all tRNA molecules
(ribothymidylate and pseudouridylate).
 Types of RNA in prokaryotes
• In eukaryotes, tRNA precursors are converted
into mature tRNAs by a series of alterations:
cleavage of a 5’ leader sequence, splicing to
remove an intron, replacement of the 3’
terminal UU by CCA and modification of
several bases.