RNA

There are three major types of RNA that participate in the process of protein synthesis: rRNA, tRNA, and mRNA.

First: rRNA:

a)     Are found in association with several proteins as components of the ribosomes, the complex structures that serve as the sites for protein synthesis.

b)    There are three distinct size species of rRNA (23S, 16S, and 5S) in prokaryotic cells while in eukaryotes there are 4 species of rRNA (28S, 18S, 5.8S, and 5S).

c)     Make up about 80% of the total RNA in the cell.

Second: tRNA:

a)     Is the smallest (4S) of the three major types of RNA molecules.

b)     There is at least one specific type of tRNA molecule for each of the 20 amino acids commonly found in proteins.

c)     Make up about 15% of the total RNA in the cell.

d)     Contain a high percentage of unusual bases (Pseudouridine), for example, dihydrouracil and have extensive intrachain base-pairing.

e)     Serves as an “adaptor” molecule that carries its specific amino acid, covalently attached to its 3ʹ-end, to the site of protein synthesis. (amino acid is attached to terminal adenine ribonucleotide at either 2’ or 3’ (OH) group.

Third: mRNA:

a)     Comprises only about 5% of the RNA in the cell.

b)     Carries genetic information from DNA for use in protein synthesis.

c)     Has special structural characteristics: a long sequence of adenine nucleotides (a poly-A “tail”) on the 3ʹ-end of the RNA chain, plus a “cap” on the 5ʹ-end consisting of a molecule of 7-methylguanosine attached through an unusual (5ʹ→5ʹ) triphosphate linkage.

d)     In eukaryotes, it undergoes changes post transcription to produce mature mRNA or heterogenous nuclear mRNA where introns which are non-coding regions are spliced and exons which are coding regions are bound together to form mature one.

Transcription process in prokaryotes:

RNA polymerase is the enzyme that is responsible for transcribing a part of DNA into mRNA in direction from 5’ to 3’ of newly forming strand.

Before we talk about the process, we will illustrate the structure of RNA polymerase.

RNA polymerase structure:

a)     Core enzyme: 2α, 1β, 1βʹ, and1Ω are part of enzyme peptide subunits where its function is 5’ to 3’ RNA polymerase activity and have no 3’ to 5’ exonuclease (proofreading capabilities).

b)     Holoenzyme: where, the s subunit (sigma factor) enables RNA pol to recognize promoter regions on DNA. The s subunit with core enzyme forms holoenzyme.

Before we illustrate steps of process, we have to explain some terms related to transcription:

1)     Sense strand is the DNA strand that is similar to mRNA. And it is in direction of 5’ to 3’.

2)     Anti-sense strand is the DNA template strand that is used by RNA pol to form mRNA. And it is in direction of 3’ to 5’.

3)     As we know that for each process there must be a start point for initiation process, so the bases to left of the start point is upstream and taking negative sign while to the right is downstream and taking positive sign.

Now: steps of mRNA synthesis include initiation, elongation and termination.

First: initiation:

In prokaryotes: Transcription begins with the binding of the RNA pol holoenzyme (sigma factor) to a region of the DNA known as the promoter, which is not transcribed.

There are some consensus sequences within promoter region that are recognized by RNA polymerase holoenzyme:

a)     -35 sequence: A consensus sequence (5-TTGACA-3), centered about 35 bases to the left of the transcription start site, is the initial point of contact for the holoenzyme.

b)     Pribnow box: A second consensus sequence (5ʹ-TATAAT-3ʹ), centered at about 10, which is the site of initial DNA melting (unwinding).

In eukaryotes: promoter has 2 characteristic sequences:

a)     CAAT sequence (-70 sequence) which is centered about 70 bases to the left of the transcription start site.

b)     Hongess box or TATA (-25 or -30 sequence) which is centered about 25 or 30 bases to the left of the transcription start site.

Second: elongation:

Once the promoter region has been recognized and bound by the holoenzyme, local unwinding of the DNA helix continues, mediated by the polymerase.

RNA pol begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made.

RNA pol uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain.

Third: Termination:

The elongation of the single-stranded RNA chain continues until a termination signal is reached. Termination can be intrinsic (spontaneous) or dependent upon the participation of a protein known as the ρ (rho) factor.

a)     ρ -independent termination: Here sequences of mRNA are self-complementary. This allows the RNA to fold back on itself, forming a GC-rich stem (stabilized by hydrogen bonds) plus a loop. This structure is known as a “hairpin. And once hairpin is formed, RNA polymerase stops the process.

b)     r -dependent termination: This requires the participation of an additional protein, rho (r) where rho binds a C-rich “rho recognition site” near the 5ʹ-end of the nascent RNA and moves along the RNA until it reaches the RNA pol paused at the termination site.

Notes:

1)     Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis. For example, rifampin (rifampicin) inhibits transcription by binding to the β subunit of prokaryotic RNA pol, and preventing chain extension (elongation).

2)     Rifampin is used in treatment of TB and box virus.

3)     Dactinomycin binds to the DNA template and interferes with the movement of RNA pol along the DNA.

After we talk about transcription in prokaryotes, we will illustrate some points of this process in eukaryotes.

Nuclear RNA polymerases of eukaryotic cells:

a)     RNA polymerase I: This enzyme synthesizes the precursor of the rRNA in the nucleolus.

b)     RNA polymerase II: This enzyme synthesizes the nuclear precursors of mRNA and small ncRNAs (non-coding mRNA), such as snRNA (regulation of gene).

c)     RNA polymerase III: This enzyme synthesizes tRNA, 5S rRNA (small subunit), and some snRNA.

Transcription process in eukaryotes:

Eukaryotic transcription involves separate polymerases for the synthesis of rRNA, tRNA, and mRNA. In addition, a large number of proteins called transcription factors (TFs) which bind to distinct sites on the DNA either within the core promoter region, close (proximal) to it, or some distance away (distal). They are required both for the assembly of a transcription complex at the promoter and the determination of which genes are to be transcribed.

Role of enhancers in gene regulation:

Are special DNA sequences that increase the rate of initiation of transcription by RNA pol II.

They can be located upstream (to the 5ʹ-side) or downstream (to the 3ʹ-side) of the transcription start site, and be close to or thousands of base pairs away from the promoter.

Inhibitors of RNA polymerase II:

α-Amanitin, a potent toxin produced by the poisonous mushroom Amanita phalloides (sometimes called “the death cap”), forms a tight complex with RNA pol II, thereby inhibiting mRNA synthesis.

Wait for us in another topic in Biochemistry!!!!!!!!