MECHANISM OF TRANSCRIPTION – THE SYNTHESIS OF RNA FROM DNA

Transcription is the process by which a specific segment of DNA is copied into RNA. This occurs by utilizing an enzyme called RNA polymerase. In eukaryotic cell, transcription takes place in nucleus whereas in prokaryotes, it takes place in cytoplasm.

Transcription is the first process in gene expression as the transcribed RNA serves to synthesize protein by a process called translation. RNA polymerase attaches to a segment of DNA called a gene. It then causes unwinding of the two strands of DNA. One of the strands is called the template or antisense strand, which is transcribed into RNA. The other strand of DNA is called the non-template or coding or strand strand as with the exception of T for U, it corresponds exactly to the sequence of the RNA primary transcript which encodes the protein.

Template strand for each gene need not necessarily be the same strand of DNA. Thus, the same strand of DNA will serve as template strand for some genes and coding strand for genes. The information in the template strand is read in 3′ – 5′ direction. The new RNA is built in 5′ – 3′ direction.

All eukaryotic cells have four major classes of RNA – ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), small RNAs (small nuclear – sn RNA and micro – mi RNA). The RNA molecules synthesized in eukaryotes are formed as precursor molecules that need to be processed into mature RNA before they can serve their function.

PROCESS OF TRANSCRIPTION

DNA dependent RNA polymerase is the enzyme that catalyzes the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene. RNA polymerase binds to a specific site – the promoter on the template strand. This is followed by initiation of RNA synthesis and the process continues till a termination sequence is encountered.

A transcription unit is defined as that region of DNA which contains the signals for transcription initiation, elongation and termination. The RNA product, which is synthesized in the 5′-3′ direction, is the primary transcript.

The starting point of transcription corresponds to the 5′ nucleotide of mRNA and is designated position +1. The positive numbers increase downstream as we go towards 3′ end. The nucleotide in the promoter adjacent to the transcription initiation site in the upstream direction is designated -1, and these negative numbers increase as the sequence proceeds upstream away from the initiation site.

RNA POLYMERASE

DNA dependent RNA polymerase is a multi-subunit enzyme that synthesizes RNA from a DNA template during transcription. It binds to template strand of DNA , unwinds it and catalyzes the formation of phosphodiester bonds to build a complementary RNA strand in the 5′ to 3′ direction.

In prokaryotes, RNA polymerase consists of a core enzyme (formed by two alpha, one beta, one beta’ , and one omega subunits) which bind to a sigma factor. Core enzyme is also termed as E. Core enzyme binds with sigma factor to form holoenzyme. The sigma subunit helps the core enzyme recognize and bind to promoter.

In eukaryotes, RNA polymerase I transcribes rRNA, RNA polymerase II transcribes mRNA, miRNA and snRNA and RNA polymerase III transcribes tRNA and 5s rRNA. Eukaryotic RNA polymerase contain two large subunits and many smaller subunits.

BACTERIAL PROMOTER

A bacterial transcription promoter is a specific DNA sequence located upstream (5′) of a gene that initiates transcription by serving as the binding site for RNA polymerase (RNAP).

COMPONENTS

• 35 base pair upstream to transcription start site (-35) : consensus sequence of 8 nucleotide pairs (5′-TGTTGACA-3′) where RNAP binds to form a closed complex.
• 10 base pair upstream (-10) : there is 6 nucleotide pair A+T rich sequence (5′-TATAAT-3′). This is known as TATA box or pribnow box. It has low melting point which helps in dissociation of two strands of DNA . This allows RNAP to gain access to nucleotide sequence immediately downstream. This combination of RNAP with promoter leads to formation of open complex.
• Spacer : The region between -35 and -10 containing 17 base pairs. It facilitates recognition of -35 and -10 sequences by RNAP.

Repressor and activator proteins lie adjacent to promoters. They enhance or inhibit pre-initiation complex (PIC) formation and transcription initiation.

Termination signals are rho factor dependent, 40 nucleotide in length. When RNAP meets termination signal, it stops, dissociates from DNA template and releases the nascent RNA transcript.

EUKARYOTIC PROMOTER

TATA box : is located 25 to 30 base pair upstream from transcription start site. It contains sequence TATAAA. It binds TATA binding protein (TBP) to initiate transcription.

Sequences of GC and CAAT located just upstream from start site determine how frequently transcription occurs.

Enhancers and repressors (silencers) – are found both upstream and downstream of transcription start site and even within genes.

Termination signals are located far downstream.

STEPS INVOLVED IN TRANSCRIPTION

The process of transcription includes – initiation, elongation and termination and processing.

INITIATION

• RNA polymerase binds to DNA and locates a promoter.
• DNA undergoes temperature dependent conformational change and the two strands unwind. This complex is termed as pre-initiation complex or PIC. The unwinding allows the active site of RNA polymerase to access the template strand.
• The first nucleotide then associates with the nucleotide binding site on the beta subunit of the enzyme. RNAP catalyzes formation of phosphodiester bond in the presence of appropriate nucleotide and a nascent RNA chain is attached to beta subunit of RNAP.
• RNAP continues to incorporate nucleotides. After adding a few nucleotides it undergoes another conformational change and moves away from promoter. This is known as promoter clearance.

ELONGATION

• The RNA transcript is extended during the elongation phase.
• RNAP reads template strand in 3′ to 5′ and writes down primary transcript in 5′ to 3′ direction. Base pairing is done according to Watson-Crick base pairing rules – G with C and A with U (as RNA contains uracil instead of thymine).
• As the elongation complex containing RNAP progresses along the DNA molecule, DNA must keep unwinding in order to provide access for appropriate base pairing. The extent of this transcription bubble (i.e, DNA unwinding) is constant throughout transcription and is about 20 base pairs per molecule of RNAP.

TERMINATION

• The termination sequence on the template strand is recognized by rho factor. When RNAP reaches the termination sequence, it separates from DNA template. RNAP then dissociates to release core enzyme and sigma factor.
• With the assistance of another sigma factor, the core enzyme then recognizes a promoter at which synthesis of a new RNA molecule commences.

PROCESSING

• Prokaryotic mRNA are subjected to little processing. Primary transcripts of prokaryotic mRNA begin to perform translation even before transcription is completed. Prokaryotic rRNA and tRNA require processing before they become functional.
• In eukaryotes, nearly all RNA primary transcripts undergo extensive processing. Processing occurs primarily within the nucleus. of cell.
• Splicing – Within a gene, the sequences that code for protein are called exons. Exons are interrupted by intervening sequences of introns which do not code for protein. Introns are removed and exons are joined together by a process called splicing. Spliceosome consists of snRNA which may join with some proteins to form snRNP. snRNPs serve to position the exons and introns for the necessary splicing reactions. Splicing starts with a cut at the junction of the 5′-exon and intron. The free 5′ terminal then forms a loop or lariat structure. A second cut is made at the junction of the intron with the 3′ exon. The lariat structure containing the intron is released and hydrolyzed. The 5′ and 3′ exons are joined to form a continuous sequence. Splicing may not necessarily involve adjacent exons. This may result in alternative splicing, leading to variability in gene expression.

• Capping – a 7-methylguanosine cap structure is added at the 5′ terminal of mRNA. The 5’cap helps to initiate the process of translation efficiently and also protects 5′ end of mRNA from attack by 5′ – 3′ exonucleases.
• Tailing – Poly (A) tails are added to the 3′ end of mRNA. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence. Enzyme poly (A) polymerase adds a poly (A) tail which is subsequently extended to as many as 200 A residues. The poly (A) tail protects 3′ end of mRNA from attack by 3′-5′ exonuclease and facilitates translation.

After the primary RNA transcript is processed in the nucleus, mature RNA is transported into the cytoplasm through nuclear pore into the cytoplasm where it is ready to perform the function of protein synthesis by translation.

DIFFERENCE BETWEEN DNA REPLICATION AND TRANSCRIPTION

• Ribonucleotides are used in transcription whereas deoxyribonucleotides are used in DNA replication.
• Thymine is replaced with uracil in RNA.
• Transcription does not require a primer as RNA polymerases can synthesize RNA de novo.
• Only portions of genome are copied into RNA during transcription, whereas the entire genome is copied during DNA replication.
• RNA polymerase lacks proofreading function.