Transcription

 the process by which RNA is synthesized from the DNA

 steps:
o initiation
o elongation
o termination

RIBONUCLEIC ACID 
Two major classes of RNA
 protein coding RNAs
1. messenger RNAs (mRNAs)
 non-protein coding RNAs
1. large noncoding RNAs
 ribosomal RNAs (rRNA)
2. long noncoding RNAs (lncRNAs)
3. small noncoding RNAs
 transfer RNAs (tRNA)
 small nuclear RNAs (snRNAs)
 micro and silencing RNAs (miRNAs and siRNAs)
 mRNAs, rRNAs and tRNAs - directly involved in protein synthesis
 SnRNAs – mRNA splicing
 mi/SiRNAs – modulation of gene expression by altering mRNA function
 lncRNAs – modulation of gene expression

Replication and Transcription

 Similarities:
o the general steps of initiation, elongation, and termination with 5′–3′ polarity
o large, multicomponent initiation complexes
o adherence to Watson–Crick base-pairing rules
 Differences:
o ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides
o U replaces T as the complementary base for A in RNA;
o a primer is not involved in RNA synthesis as RNA polymerases have the ability to initiate synthesis de novo
o in a given cell only portions of the genome are vigorously transcribed or copied into RNA, whereas the entire
genome must be copied, once and only once during DNA replication
o there is no highly active, efficient proofreading function during RNA transcription.

Template strand and Coding Strand

 Template strand
o strand that is transcribed or copied into an RNA molecule
o read out in the 3′–5′ direction
o primary transcript is complementary to the template stand
 Coding strand
o non template strand
o nucleotide sequence of an RNA transcript will be the same (except for U replacing T) as that of the coding strand.
Promoters
 Bacterial Promoters
5′-TGTTGACA-3′
o 35-bp upstream of the transcription start site

TATA Box (5′-TATAAT-3′)

o 10 nucleotides upstream of the transcription start site
o has a lower melting temperature because of its lack of GC nucleotide pairs

Transcription unit
 region of DNA that includes the signals for transcription initiation, elongation, and termination.

Primary transcript
 RNA product, which is synthesized in the 5′-3′ direction
 The 5′ termini of the primary RNA transcript and the mature cytoplasmic RNA are identical

RNA polymerase
 DNA-Dependent RNA Polymerase
 enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the
gene
 The enzyme attaches at a specific site—the promoter— on the template strand.
 This is followed by initiation of RNA synthesis at the starting point, and the process continues until a termination sequence is
reached

Bacterial DNA-Dependent RNA Polymerase

 Multisubunit enzyme
 Holoenzyme = core enzyme + σ subunit
core enzyme
o two identical α subunits
o two large β and β′ subunits
o ω subunit.
β subunit
o binds Mg2+ ions and composes the catalytic subunit
σ subunit
o enables the core enzyme to recognize and bind the promoter region to form the preinitiation complex (PIC).
o σ association with core RNA polymerase decreases its affinity for non-promoter DNA while simultaneously
increasing holoenzyme affinity for promoter DNA.

Eukaryotic RNA Polymerase

 DNA-dependent RNA polymerases responsible for transcription of different sets of genes
 exhibit more complex subunit profiles than prokaryotic RNA polymerases.
 They all have two large subunits, which remarkably bear strong sequence and structural similarities to prokaryotic β and β′
subunits, and a number of smaller subunits

Topoisomerase
 both precedes and follows the progressing RNA
polymerase to prevent the formation of superhelical
tensions that would serve to increase the energy required to
unwind the template DNA ahead of RNAP.

Enhancers and Repressors

  Certain DNA elements facilitate or enhance initiation at the promoter and hence are termed enhancers and those which
repress initiation at the promoter are called repressors or silencers.

Nucleosome = DNA + Histone

Chromatin
 Euchromatin
o relaxed form of chromatin
o site of most active segments
 Heterochromatin
o condensed
o site of most inactive segments
 Chromatin remodeling
o interconversion between euchromatin and heterochromatin

TRANSCRIPTION
Tempate binding and closed RNA polymerase-promoter complex formation:
 RNA polymerase binds, with low affinity, to many regions of DNA, but it scans the DNA sequence until it recognizes
promoter regions of DNA to which it binds with higher affinity.

Open promoter complex formation:

 Once the process of strand separation occurs, the combination of RNA polymerase plus promoter is called the open complex.
This complex is also referred to as the pre-initiation complex or PIC.
 Strand separation allows the polymerase to access the coding information in the template strand of DNA.

Chain initiation:
 Using the coding information of the template RNAP catalyzes the coupling of the first base (often a purine) to the second, to
form a dinucleotide.
Promoter clearance:
 After RNA chain length reaches ~10–20 nucleotides,
 the polymerase undergoes a conformational change, then it moves away from the promoter, transcribing down the
transcription unit.

Chain elongation:
 Successive residues are added to the 3’ –OH terminus of the nascent RNA molecule until a transcription termination signal
(T) is encountered.

Chain termination and RNAP release:

 By two methods:

 rho factor dependent termination

o Termination signal for transcription in the template strand is identified by r factor.
o rho factor is an ATP dependent RNA-DNA helicase that disrupts the nascent RNA-DNA complex.
 Intrinsic (Spontaneous) or r (rho) factor independent termination
o RNA polymerase identifies the termination signal on the template strand without the aid of r factor.
o But for this termination the nascent RNA should have certain pre-requisites.
 GC rich region that forms a hairpin turn
 U rich region after the GC rich region. The binding of A-U is weak, hence, facilitate termination of
transcription.