RNA Synthesis

Synthesis of RNA

Transcription refers to RNA synthesis from DNA. The enzyme DNA dependent RNA polymerase catalyses this process. Transcriptional process is very similar in prokaryotes and eukaryotes given what is known about eukaryotic transcription so far. Transcription can be sub-divided into 3 stages

• Initiation
• Elongation and
• Termination

These stages occur in a cyclical process in prokaryotes.

Initiation of Transcription

The initiation stage is the most understood stage of all the stages in prokaryotes it begins with the binding of RNA Polymerase to the promoter region of the gene to be transcribed, RNA polymerase is guided by a sigma factor which is usually bound to the enzyme complex. Sigma (σ) factor recognizes certain consensus sequence rich in adenine and thymine residues (TATAA) within the promoter region for correct binding. Binding of the RNA polymerase and sigma factor to the promoter region of the gene leads to the formation of the pre-initiation complex (PIC). In eukaryotes formation of the pre-initiation complex is far more complex and involves more transcription factors in association with RNA polymerase II. Most mammalian genes have a TATA box that is usually located 25–30 bp upstream from the transcription start site. The consensus sequence for a TATA box is TATAAA, though numerous variations have been characterrized. The TATA box is bound by 34 kDa TATA binding protein (TBP), which in turn binds several other proteins called TBP-associated factors (TAFs). This complex of TBP and TAFs is referred to as TFIID. Binding of TFIID to the TATA box sequence is believed to represent the first step in the formation of the transcription complex on the promoter.  Initiation ends when DNA dependent RNA polymerase coubles the first ribonucleotide to the second incoming ribonucleotide via the formation of a phosphodiester linkage.

culled from helicasepbworks.com

Elongation

Elongation of the nascent RNA transcript occurs with the addition of successive rNTPs to the 3’ end of the growing RNA chain for elongation to occur, the DNA double helix must be partially unwound for access to the correct base pairing. Usually a transcription bubble consisting of 15-20 nucleotides long is formed. DNA dependent RNA polymerase catalyses polymerization of the incoming rNTP to the growing RNA in a specific sequence dictated by the template strand with realease of pyrophosphate which is further hydrolysed to Pi rendering the entire process irreversible.

Termination

The termination of the synthesis of the RNA molecule in prokaryotes 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. After termination

of synthesis of the RNA molecule, the enzyme separates from the DNA template and probably dissociates

to free the core enzyme and free σ factor.

Rho-dependent transcription termination signals in E coli also appear to have a distinct consensus sequence. The conserved consensus sequence, which is about 40 nucleotide pairs contains a hyphenated or interrupted inverted repeat followed by a series of AT base pairs.

Post Transcriptional Modifications

At the end of transcription of in eukaryotes, the mRNA transcript is immediately capped at the 5' end with a 7-methyl guanosine cap- this confers protection on the RNA molecule from digestion by the organisms own nuclease enzymes a further post-transcriptional modification is the addition of a poly A tail to the 3' end of the RNA molecule.
The mRNA transcript synthesized by RNA polymerase II is modified in eukaryotes IS undergoes the folllowing modifications.

5’ Capping (7-methylguanosine cap)

3’ polyadenylation

Intron Removal and Exon Splicing

Nascent RNA transcripts from protein-coding genes are called hnRNA (pre-mRNA) . They are associated with proteins until they are exported into the cytosol. 

hnRNP is thought to Prevent folding of pre-mRNA into secondary structures that may inhibit its interactions with other proteins. May associate with the splicing apparatus. Transport of mRNA out of the nucleus.
5’ CAPPING

After the mRNA transcript is about 25-30 nucleotides long a 7-methylguanosine is added to the 5’ end. The capping enzyme associates with the phosphorylated CTD of RNA polymerase II. Protects the 5’ from enzymatic degradation in the nucleus and cytoplasm by ribonucleases and assists in export to the cytosoplasm.

3’POLYADENYLATION

 at the end of synthesis of the primary transcript, a poly A tail is added to the free 3’ OH. Up to 250 Adenylate residues may be added. Carried out by poly(A) polymerase, this protects the mRNA from degradation from the 5’ end.

RNA SPLICING

unlike prokaryotic genes, eukaryotic genes are ususlly interupted by introns (non-coding sequences)Introns are removed and the exons are spliced together by the spliceosome complex. Splice sites occur at both the 5’ and 3’ ends of introns. The intron is cut at the splice sites. Only 30-40 nucleotides at each end of an intron are required for splicing.

The strand that is transcribed or copied into an RNA molecule is referred to as the template strand (+strand) of the DNA. The other DNA strand is frequently referred to as the coding strand of that because it is entirely similar in sequence to the RNA strand but for the substitution of U for T.