Transcription-Lecture-1

RNA Synthesis

The synthesis of an RNA molecule from DNA is called Transcription. All eukaryotic cells have five major classes of RNA: ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), and small nuclear RNA and microRNA (snRNA and miRNA). The first three are involved in protein synthesis, while the small RNAs are involved in mRNA splicing and gene regulation.

Similarities between Replication and Transcription

The processes of DNA and RNA synthesis are similar in that they involve

(1) the general steps of initiation, elongation, and termination with 5′ to 3′ polarity;

(2) large, multicomponent initiation complexes; and

(3) adherence to Watson-Crick base-pairing rules.

Differences between Replication and Transcription

(1) Ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides;

(2) U replaces T as the complementary base pair for A in RNA;

(3) A primer is not involved in RNA synthesis;

(4) Only a portion of the genome is transcribed or copied into RNA, whereas the entire genome must be copied during DNA replication; and

(5) There is no proofreading function during RNA transcription.

Template strand

• The sequence of ribonucleotides in an RNA molecule is complementary to the sequence of deoxyribonucleotides in one strand of the double-stranded DNA molecule.
• The strand that is transcribed or copied into an RNA molecule is referred to as the template strand of the DNA.
• The other DNA strand, the non-template strand, is frequently referred to as the coding strand of that gene.
•  It is called this because, with the exception of T for U changes, it corresponds exactly to the sequence of the RNA primary transcript, which encodes the (protein) product of the gene.
•  In the case of a double-stranded DNA molecule containing many genes, the template strand for each gene will not necessarily be the same strand of the DNA double helix.
• Thus, a given strand of a double-stranded DNA molecule will serve as the template strand for some genes and the coding strand of other genes.
• The information in the template strand is read out in the 3′ to 5′ direction.

Transcription unit

• DNA-dependent RNA polymerase is the 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 the initiation of RNA synthesis at the starting point, and the process continues until a termination sequence is reached (Figure-3).
• A transcription unit is defined as that region of DNA that includes the signals for transcription initiation, elongation, and termination.

Primary transcript

• The RNA product, which is synthesized in the 5′ to 3′ direction, is the primary transcript.
• In prokaryotes, this can represent the product of several contiguous genes
•  In mammalian cells, it usually represents the product of a single gene
•  The 5′ terminals of the primary RNA transcript and the mature cytoplasmic RNA are identical.
• The starting point of transcription corresponds to the 5‘ nucleotide of the mRNA.
• This is designated position +1, as is the corresponding nucleotide in the DNA (Figure-2.3)
• The numbers increase as the sequence proceeds downstream.
• The nucleotide in the promoter adjacent to the transcription initiation site is designated -1,
•  These negative numbers increase as the sequence proceeds upstream, away from the initiation site.
• This provides a conventional way of defining the location of regulatory elements in the promoter.

Bacterial DNA-Dependent RNA Polymerase- (figure-1)

• The DNA-dependent RNA polymerase (RNAP) of the bacterium Escherichia coli exists as an approximately 400 kDa core complex consisting of-
• two identical α subunits,
• similar but not identical β andβ ‘ subunits, and
• an ω  subunit.
• Beta is thought to be the catalytic subunit.
• RNAP, a metalloenzyme, also contains two zinc molecules.
• The core RNA polymerase associates with a specific protein factor (the sigma σ factor) that helps the core enzyme recognize and bind to the specific deoxynucleotide sequence of the promoter region to form the preinitiation complex (PIC) (Figure-2)
•  Bacteria contain multiple factors, each of which acts as a regulatory protein.

Figure-1- showing the subunits of bacterial RNA polymerase

Mammalian cells possess three distinct nuclear DNA-Dependent RNA Polymerases

• RNA polymerase I is for the synthesis of r RNA
• RNA polymerase II is for the synthesis of m RNA and miRNA
• RNA polymerase III is for the synthesis of tRNA/5S rRNA, snRNA

Steps in RNA (Prokaryotic transcription)

The process of transcription of a typical gene of E.Coli can be divided into three phases-

i) Initiation

ii) Elongation

iii) Termination

i) Initiation

• Initiation of transcription involves the binding of the RNA polymerase holoenzyme to the promoter region on the DNA to form a preinitiation complex, or PIC(Figure-2)

 

Figure-2- showing the promoter identification by sigma subunit of RNA Polymerase.

• Characteristic “Consensus” nucleotide sequences of the prokaryotic promoter region are highly conserved.

Structure of bacterial prokaryotic promoter region

• Pribnow box

This is a stretch of 6 nucleotides ( 5′- TATAAT-3′) centered about 8-10 nucleotides to the left of the transcription start site.(Figure-3)

•  -35 Sequence

A second consensus nucleotide sequence ( 5′- TTGACA-3′), is centered about 35 bases to the left of the transcription start site (figure-3).

Figure-3-Bacterial promoters share two regions of the highly conserved nucleotide sequences. These regions are located 35 and 10 bp upstream (in the 5′ direction of the coding strand) from the start site of transcription, which is indicated as +1. By convention, all nucleotides upstream of the transcription initiation site (at +1) are numbered in a negative sense and are referred to as 5′-flanking sequences. Also by convention, the DNA regulatory sequence elements (TATA box, etc) are described in the 5′ to 3′ direction and as being on the coding strand. These elements function only in double-stranded DNA.

• Binding of RNA-polymerase (RNAP) to the promoter region is followed by a conformational change of the RNAP, and the first nucleotide (almost always a purine) then associates with the initiation site on the subunit of the enzyme.
• In the presence of the appropriate nucleotide, RNAP catalyzes the formation of a phosphodiester bond, and the nascent chain is now attached to the polymerization site on the subunit of RNAP.
•  In both prokaryotes and eukaryotes, a purine ribonucleotide is usually the first to be polymerized into the RNA molecule.
• After 10–20 nucleotides have been polymerized, RNAP undergoes a second conformational change leading to promoter clearance.
•  Once this transition occurs, RNAP physically moves away from the promoter, transcribing down the transcription unit, leading to the next phase of the process, elongation.

II) Elongation

• As the elongation complex containing the core RNA polymerase progresses along the DNA molecule, DNA unwinding must occur in order to provide access for the appropriate base pairing to the nucleotides of the template strand.
•  The extent of this transcription bubble (ie, DNA unwinding) is constant throughout and is about 20 base pairs per polymerase molecule. (Figure-4)
• RNA polymerase has associated with it an “unwindase” activity that opens the DNA helix.
• Topoisomerase both precedes and follows the progressing RNAP to prevent the formation of superhelical complexes.

iii)  Termination

Termination of the synthesis of the RNA molecule in bacteria is of two types-

a) Rho (ρ) dependent termination (figure-4)

• The termination process is signaled by a sequence in the template strand of the DNA molecule—a signal that is recognized by a termination protein, the rho (ρ) factor.
• Rho is an ATP-dependent RNA-stimulated helicase that disrupts the nascent RNA-DNA complex.

Figure-4- showing the process of transcription. The promoter region is identified by the sigma factor of RNA polymerase. The strand unwinding continues with the elongation phase, the base-pairing rule is followed until there is a signal for the termination of transcription. Rho-dependent termination is shown in the figure.

b) Rho-independent termination

• This process requires the presence of intrachain self-complementary sequences in the newly formed primary transcript so that it can acquire a stable hairpin turn that slows down the progress of the RNA polymerase and causes it to pause temporarily.
• Near the stem of the hairpin, a sequence occurs that is rich in G and C. This stabilizes the secondary structure of the hairpin (figure-5).

Figure-5-showing the hairpin structure formed in the primary  transcript structure due to the presence of self-complementary base pairs

• Beyond the hairpin, the RNA transcript contains a string of Us,(Figure-3) the bonding of Us to the corresponding As is weak. This facilitates the dissociation of the primary transcript from DNA (Figure-6).

Figure-6- showing the process of transcription. A, B and C showing initiation, elongation, and termination respectively.

After termination of the synthesis of the RNA molecule, the enzyme separates from the DNA template. With the assistance of another factor, the core enzyme then recognizes a promoter at which the synthesis of a new RNA molecule commences.