RNA: Structures, types, transcription, control at initiation , control at termination, attenuation, heterogenous RNA processing, capping and tailing

Structure and Types of RNA:-

Basic Structure of RNA:- The ribonucleic acid has all the components same to that of the DNA with only 2 main differences within it-

i. Thymine which is replaced by the uracil. 

ii. Deoxyribose sugar is replaced by Ribose sugar.

Types of RNA:- 

i. mRNA:- It is transcribed from DNA and contains the genetic information to make proteins. It has a start codon and a stop codon for protein synthesis. Prokaryotic mRNA is simple. Eukaryotic mRNA has a 5' cap and 3' poly A tail. The 5’ cap protects the mRNA from degradation, and the 3’ poly(A) tail contributes to the stability of mRNA and aids it in transport. 

ii. tRNA:- These are RNA molecules that translate mRNA into proteins. They of a 3’ acceptor site, 5’ terminal phosphate, D arm, T arm, and anticodon arm. The primary function of a tRNA is to carry amino acids on its 3’ acceptor site to a ribosome complex with the help of aminoacyl-tRNA synthetase. Aminoacyl-tRNA synthetases are enzymes that load the appropriate amino acid onto a free tRNA to synthesize proteins. Once an amino acid is bound to tRNA, the tRNA is considered an aminoacyl-tRNA. tRNA has an L shaped 3D structure. It is specific to each amino acid and carries an amino acid to the growing chain of a polypeptide during the translation process.

Secondary Structure:- It has a shape similar to Clover leaf.

Tertiary Structure:- It has a shape similar to Inverse L letter.

iii. rRNA:- rRNA forms ribosomes, which are essential in protein synthesis. A ribosome contains a large and small ribosomal subunit. In prokaryotes, a small 30S and large 50S ribosomal subunit make up a 70S ribosome. In eukaryotes, the 40S and 60S subunit form an 80S ribosome. The ribosomes contain an exit (E), peptidyl (P), and acceptor (A) site to bind aminoacyl-tRNAs and link amino acids together to create polypeptides. 

iv. Small nuclear RNAs (snRNA):- These are non-coding RNAs that are responsible for splicing introns. The snRNAs join with proteins to form small nuclear ribonucleoproteins (snRNP), which most commonly contain U1, U2, U4, U5, and U6 snRNA molecules. 

v. MicroRNA (miRNA):- These are non-coding RNAs mainly involved in gene regulation. They are mostly processed from introns and are transcribed into primary miRNA from the host gene by RNA polymerase II. 

vi. Small Interfering RNAs (siRNA):- These are double-stranded, non-coding RNAs that inhibit gene expression through RNA interference. They interfere with gene expression by degrading mRNA and preventing the translation of proteins.

Transcription:-

Introduction:-

•   It is the process of copying genetic information from one strand of the DNA into RNA.

•   Here, adenine pairs with uracil instead of thymine.

•   Both strands are not copied during transcription, because:-

-   The code for proteins is different in both strands. This complicates the translation.

-   If 2 RNA molecules are produced simultaneously this would be complimentary to each other, hence form a double stranded RNA. This prevents translation.

Transcription Unit:-

•   It is the segment of DNA between the sites of initiation and termination of transcription. It consists of 3 regions:-

-   A promoter (Transcription start site):- Binding site for RNA polymerase.

-   Structural gene:- The region between promoter and terminator where transcription takes place.

-   A terminator:- The site where transcription stops.

•   The DNA- dependent RNA polymerase catalyzes the polymerization only in 5’→3’direction.

•   3’→5’ acts as template strand and 5’→3’ acts as coding strand.

3’-ATGCATGCATGCATGCATGCATGC-5’ template strand.

5’-TACGTACGTACGTACGTACGTACG-3’ coding strand.

Transcription unit and gene:-

•   Gene:- Functional unit of inheritance. It is the DNA sequence coding for RNA molecule.

•  Cistron:- A segment of DNA coding for a polypeptide.

•  Structural gene in a transcription unit is of 2 types:-

i. Monocistronic structural genes (split genes):- It is seen in eukaryotes. Here, the coding sequences  exons are interrupted by introns.

ii. Polycistronic structural genes:- It is seen in prokaryotes. Here, there are no split genes.

Reverse Transcription:- It is the process in cells by which an enzyme makes a copy of DNA from RNA. The enzyme that makes the DNA copy is called reverse transcriptase and is found in retroviruses, such as the human immunodeficiency virus (HIV).

Steps of transcription in prokaryotes:-

i. Initiation:- Here, the enzyme RNA polymerase binds at the promoter site of DNA. This causes the local unwinding of the DNA double helix. An initiation factor (σ factor) present in RNA polymerase initiates the RNA synthesis.

ii. Elongation:- The RNA chain is synthesized in the 5’-3’ direction. In this process, activated ribonucleoside triphosphates (ATP, GTP, UTP & CTP) are added. This is complementary to the base sequence in the DNA template.

iii. Termination:- A termination factor (ρ factor) binds to the RNA polymerase and terminates the transcription.

NOTE:- In bacteria (Prokaryotes) transcription and translation can be coupled (Translation can begin before mRNA is fully transcribed) because:-

-   mRNA requires no processing to become active.

-   Transcription and translation take place in the same compartment (no separation of cytosol and nucleus).

Polysome or Polyribosome or Ergosome:- It is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides.

Eukaryotic transcription:- 

In eukaryotes, there are 2 additional complexities-

i. There are 3 RNA polymerases:-

•  RNA polymerase I:- Transcribes rRNAs (28S, 18S & 5.8S).

•  RNA polymerase II:- Transcribes mRNA.

•  RNA polymerase III:- Transcribes tRNA, 5S rRNA and snRNAs.

ii. The primary transcripts (hnRNA):- It contain both the exons and introns and is non-functional. Hence introns have to be removed. For this, it undergoes splicing process.

Control at Initiation:-

i. Steric hindrance for RNA polymerase binding:- If the operator and – 35 sequence overlap, repressor could prevent the formation of a closed complex by preventing polymerase binding. It is also possible that repressor binding can prevent the activator's interaction with polymerase and/or DNA.

ii. Inhibition of open complex formation:- Isomerization is a step in which RNA polymerase conformation is changed and a more stable complex is formed spanning the region of + 5 to beyond – 35 sequence. The exact boundary of nucleotides protected by RNA polymerase in footprinting

experiments varies depending on the promoter used in an experiment. The repressor when bound at or near the – 10 region of the promoter (the site of complex formation) could distort the DNA and as a consequence, formation of stable open complexes may be prevented. The Arc repressor involved in bacteriophage P22 lysogeny is believed to repress the Pant promoter in this fashion.

iii. Inhibition of initiation complex formation:- RNA polymerase initiates RNA synthesis with the formation of nascent RNA of 3-8 nucleotides. During this stage, polymerase is still bound to the promoter. The repressor which is bound to the operator may make direct contacts with the enzyme present in open complex state, thus preventing the subsequent step, i.e. oligoribonucleotide formation. Studies with the gal repressor support this mechanism. The gal repressor does not block open complex formation at the gal operon promoter and hence is believed to interfere at the stage of oligoribonucleotide formation.

iv. Increased abortive initiation:- Once an initiation complex is formed, RNA polymerase moves out of the promoter and gets locked into the elongation mode. The repressor could make contacts with RNA polymerase and block promoter clearance. 

Control at termination:-

Attenuation:- Attenuation is a regulatory mechanism used in bacterial operons to ensure proper transcription and translation. In bacteria, transcription and translation are capable of proceeding simultaneously. The need to prevent unregulated and unnecessary gene expression can be prevented by attenuation, which is characterized as a regulatory mechanism.

i. Transcriptional-attenuation:- It is characterized by the presence of an attenuator within the DNA sequence that results in formation of mRNA-stem loops that prevent further transcription from occurring. The non-functional RNA produced prevents proper transcription.

ii. Translational-attenuation:- It is characterized by the misfolding of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence, responsible for ribosomal binding to allow proper translation, is inaccessible because it is folded into a hairpin-loop structure, thus, translation cannot occur.

Heterogenous RNA Processing:- The hnRNA is the collective term for the unprocessed mRNA (pre-mRNA) molecules in the nucleus. It contain both the exons and introns and is non-functional. Hence introns have to be removed. For this, it undergoes the following processes-

i. Splicing:- From hnRNA introns are removed by the spliceosome and exons are joined together.

ii. Capping:- Here, a nucleotide methyl guanosine triphosphate (cap) is added to the 5’ end of hnRNA.

iii. Tailing (Polyadenylation):- Here, adenylate residues (200-300) are added at 3’-end. It is the fully processed hnRNA, now called mRNA.