RNA and Protein Synthesis

Overview of the Central Dogma

The central dogma of molecular biology describes the flow of genetic information from DNA to RNA to protein. This process is fundamental to all living organisms and underlies the mechanisms of gene expression and regulation.

• DNA serves as the template for RNA synthesis (transcription).

• RNA carries genetic information and is translated into proteins (translation).

• Proteins are the functional molecules responsible for cellular structure and activity.

Types and Functions of RNA

Ribosomal RNA (rRNA)

rRNA is a structural and functional component of ribosomes, facilitating protein synthesis by providing a platform for mRNA and tRNA interaction.

• Prokaryotic ribosomes contain 5S, 16S, and 23S rRNA.

• Eukaryotic ribosomes contain 5S, 5.8S, 18S, and 28S rRNA.

• rRNA sequences are complementary to regions of mRNA, aiding in translation.

Transfer RNA (tRNA)

tRNA acts as an adaptor molecule, carrying amino acids to the ribosome and matching them to the appropriate codons in mRNA during translation.

• Has a cloverleaf secondary structure and a complex 3D tertiary structure.

• Contains modified bases such as dihydrouridine (DHU), ribosylthymine (rT), pseudouridine (Ψ), and inosine (I).

• 3'-end (acceptor stem) is the site of amino acid attachment.

Other Non-coding RNAs

Non-coding RNAs do not encode proteins but play essential roles in gene regulation and RNA processing.

• snRNA: Involved in splicing.

• snoRNA: rRNA processing.

• miRNA & siRNA: Inhibit gene expression by binding to target mRNA sequences.

• piRNA: Repression of transposons and chromatin modification.

• lncRNA: Regulates gene expression.

Messenger RNA (mRNA)

mRNA carries genetic information from DNA to the ribosome for protein synthesis.

• Prokaryotic mRNAs are polycistronic (carry information for several polypeptides).

• Eukaryotic mRNAs are monocistronic (one polypeptide per mRNA), contain introns and exons, and undergo post-transcriptional modifications.

• Initiation codon: AUG; Termination codons: UAG, UGA, UAA.

Enzymatic Synthesis of RNA (Transcription)

RNA Synthesis Mechanism

RNA is synthesized from ribosyl-NTPs (A, G, C, U) by RNA polymerase, which does not require a primer. The RNA chain grows in the 5' to 3' direction, and the RNA strand is antiparallel to the DNA template strand.

• Phosphodiester bond formation between 3'-OH and 5'-P.

• 5'-end of RNA has a triphosphate group.

Transcription in Eukaryotes

Eukaryotic transcription involves three RNA polymerases, each responsible for different types of RNA.

• Pol I: rRNA (nucleolus), insensitive to amanitin.

• Pol II: mRNA and snRNA, strongly inhibited by amanitin.

• Pol III: tRNA, 5S rRNA, inhibited by high amanitin concentration.

• Promoters include TATA box and CAAT box.

• Many non-polymerase factors are required for enzyme binding to DNA.

Post-Transcriptional Modifications of mRNA

Eukaryotic mRNA Processing

Newly synthesized mRNA undergoes several modifications to become mature and functional.

• 5' capping: Addition of 7-methylguanosine to protect mRNA from degradation.

• Polyadenylation: Addition of a poly(A) tail at the 3' end, signaled by AAUAAA sequence.

• Splicing: Removal of introns and joining of exons by the spliceosome (complex of snRNAs and proteins).

• Alternative splicing and RNA editing allow for the production of modified proteins.

RNA Editing Example: Apolipoprotein B

RNA editing can change the protein product by modifying specific nucleotides in the mRNA.

• Human APO-B gene: Codon 2153 (CAA) encodes glutamine (Gln).

• In the intestine, cytidine deaminase converts CAA to UAA (stop codon), producing a truncated protein.

Translation: Protein Synthesis

The Genetic Code

The genetic code consists of 64 codons, each specifying an amino acid or a start/stop signal. It is nearly universal among organisms.

• Sixty-one codons correspond to 20 amino acids (degeneracy).

• AUG: Start codon (Met); GUG: Rarely used as start.

• UAA, UAG, UGA: Stop codons.

• Exceptions exist in mitochondrial genetic code.

Initiation of Translation in Prokaryotes and Eukaryotes

Translation initiation involves the assembly of the ribosome, mRNA, and initiator tRNA. Differences exist between prokaryotic and eukaryotic systems.

• Prokaryotes: Coupled transcription and translation; polycistronic mRNA.

• Eukaryotes: Monocistronic mRNA; transcription and translation are separated.

Post-Translational Modifications of Proteins

Types of Modifications

Proteins undergo various modifications after translation to become fully functional.

• Deformylation and removal of N-terminal amino acids.

• Acylation, methylation, hydroxylation, carboxylation, phosphorylation.

• Attachment of prosthetic groups (e.g., heme, biotin).

• Glycosylation and disulfide bond formation.

Clinical Disorders Related to Post-Translational Modifications

• Mucolipidosis II (I-cell disease): Mislocalization of lysosomal enzymes due to lack of mannose-6-phosphate.

• α1-antitrypsin deficiency: Defects in secretion, leading to lung and liver damage.

Inhibitors of Protein Synthesis: Antibiotics

Mechanisms of Inhibition

Various antibiotics inhibit protein synthesis by targeting different steps in translation.

• Aminoglycosides: Prevent correct initiation by interfering with formyl-tRNA binding.

• Puromycin: Causes premature chain termination.

• Tetracyclines: Inhibit aminoacyl-tRNA binding to the 30S subunit.

• Macrolides: Inhibit peptide bond formation and translocation.

• Lincosamides: Interfere with aminoacyl end binding and destabilize ribosomes.

• Chloramphenicol: Inhibits peptidyl transferase activity.

Inhibitors of Protein Synthesis in Eukaryotes

Some inhibitors specifically target eukaryotic ribosomes or mitochondrial ribosomes.

Collagen Biosynthesis and Disorders

Collagen Structure and Biosynthesis

Collagen is the primary structural protein in animal extracellular matrices, providing strength and elasticity.

• Basic unit: Tropocollagen, composed of three polypeptide chains (Gly-X-Y repeats).

• Post-translational modifications: Hydroxylation, glycosylation, folding, conversion to collagen, self-assembly, oxidative deamination.

• Intracellular steps: Synthesis and assembly of procollagen.

• Extracellular steps: Transformation to collagen and incorporation into fibrils.

Collagen Disorders

• Ehlers-Danlos Syndrome (EDS): Connective tissue disorder with multiple types, caused by defects in collagen synthesis or processing.

• Scurvy: Vitamin C deficiency leading to defective collagen and blood vessels.

• Epidermolysis bullosa simplex: Glycosylation defect in collagen.

• Osteogenesis Imperfecta: Disorder of type I collagen, caused by mutations in COL1A1 or COL1A2.

• Lathyrism: Inactivation of lysyl oxidase, affecting collagen and elastin tissues.

• Marfan syndrome: Mutation in fibrillin-1 gene, affecting collagen crosslinking.

• Menkes’ syndrome: Deficiency in copper-containing enzymes, affecting collagen and other tissues.