biochem final

Nucleic Acid Structure and Function

DNA and RNA are both nucleic acids made of nucleotide monomers, each containing a nitrogenous base, a sugar, and a phosphate group. DNA uses deoxyribose, which lacks a hydroxyl group on the 2’ carbon of the sugar, while RNA uses ribose, which has an –OH group at that position. This difference makes RNA more chemically reactive and less stable than DNA, which is beneficial for its roles in gene expression rather than long-term information storage.

Both DNA and RNA are polynucleotides, meaning their nucleotides are linked together by phosphodiester bonds between the 3’ hydroxyl and 5’ phosphate groups. These chains have directionality: the 5’ end has a free phosphate group, and the 3’ end has a free hydroxyl group. This polarity is critical for replication and transcription.

The nitrogenous bases are divided into purines (adenine and guanine) and pyrimidines (cytosine, thymine, and uracil). In DNA, adenine (A) pairs with thymine (T) via two hydrogen bonds, and guanine (G) pairs with cytosine (C) via three hydrogen bonds. In RNA, uracil (U) replaces thymine and pairs with adenine.

DNA exists as a double helix with antiparallel strands: one runs 5’ to 3’, the other 3’ to 5’. This helical structure is stabilized by base pairing and base stacking. RNA is usually single-stranded but folds into complex secondary structures (e.g., stem-loops, bulges, hairpins) due to internal base pairing, which is essential for its catalytic and structural roles.

DNA Encodes Genetic Information

DNA holds the genetic blueprint of all cells. Although every cell in an organism contains the same DNA, different cell types express different genes based on their function — this is differential gene expression.

A gene is defined as a sequence of DNA that codes for a functional product, either a protein or a functional RNA. In prokaryotes, genes are often organized into operons, allowing multiple genes to be transcribed from a single promoter into a polycistronic mRNA. In eukaryotes, each gene is typically transcribed individually into monocistronic mRNA, and genes contain introns (non-coding regions) and exons (coding regions). Introns must be removed during mRNA processing before translation.

DNA Replication

DNA replication is semiconservative, meaning each new DNA molecule consists of one parental strand and one newly synthesized strand. Replication begins at specific origins of replication and proceeds bidirectionally from each origin, forming two replication forks.

Because DNA polymerases can only add nucleotides to a free 3’ –OH group, replication occurs 5’ to 3’. The leading strand is synthesized continuously in the direction of the fork. The lagging strand is synthesized discontinuously, forming short fragments called Okazaki fragments, which are later joined by DNA ligase.

The process involves several key enzymes:

• Helicase unwinds the double helix.

• Topoisomerase relieves torsional strain ahead of the fork.

• Primase synthesizes RNA primers to initiate synthesis.

• DNA polymerase III extends the DNA strand from the primer.

• DNA polymerase I replaces RNA primers with DNA.

• Ligase seals the nicks between Okazaki fragments.

DNA polymerases also have 3’ to 5’ exonuclease activity for proofreading, reducing replication errors.

Some DNA viruses (like HPV and SV40) use host replication machinery. SV40 encodes a large T antigen that acts as its helicase, recruiting host DNA polymerase to replicate its genome.

Transcription

Transcription is the synthesis of RNA from a DNA template. It is performed by RNA polymerase, which does not require a primer, and proceeds in the 5’ to 3’ direction, reading the template strand in the 3’ to 5’ direction.

In prokaryotes, transcription and translation occur simultaneously in the cytoplasm. RNA polymerase binds directly to DNA promoters with the help of a σ factor, which helps initiate transcription.

In eukaryotes, transcription occurs in the nucleus and involves three RNA polymerases:

• RNA Pol I: synthesizes most rRNA.

• RNA Pol II: synthesizes mRNA and some snRNA.

• RNA Pol III: synthesizes tRNA, 5S rRNA, and other small RNAs.

RNA polymerase II requires general transcription factors (GTFs) to initiate transcription at a core promoter (often including a TATA box). Transcription initiation forms a pre-initiation complex (PIC), including TFIIH, which unwinds DNA and phosphorylates the CTD (C-terminal domain) of Pol II, allowing transcription to begin.

Once transcription begins, the mRNA transcript undergoes co-transcriptional processing:

• 5’ capping (addition of a 7-methylguanosine cap).

• Splicing (removal of introns by the spliceosome).

• 3’ polyadenylation (addition of a poly-A tail).

These modifications are essential for mRNA stability, export, and translation.

Translation

Translation is the process of synthesizing a protein from an mRNA template, carried out by ribosomes in the cytoplasm.

Ribosomes are made of rRNA and proteins and have three sites:

• A site (Aminoacyl): where incoming tRNA binds.

• P site (Peptidyl): where the growing peptide chain is held.

• E site (Exit): where tRNAs leave the ribosome.

Translation proceeds in three phases:

Initiation:
In prokaryotes, the small ribosomal subunit binds to the Shine-Dalgarno sequence on the mRNA. In eukaryotes, the small subunit binds the 5’ cap and scans for the start codon (AUG), often within a Kozak sequence. The initiator tRNA, carrying methionine, binds the start codon, and the large subunit joins.

Elongation:
Each new tRNA brings an amino acid to the A site. A peptidyl transferase activity in the large subunit forms a peptide bond between the amino acids in the P and A sites. The ribosome translocates, shifting the tRNAs and making room for the next aminoacyl-tRNA. This process requires GTP and elongation factors.

Termination:
When a stop codon (UAA, UAG, UGA) enters the A site, release factors bind and trigger hydrolysis of the bond between the polypeptide and the tRNA in the P site, releasing the completed protein.

tRNAs play a crucial role. Each tRNA has an anticodon that base-pairs with a codon on the mRNA, and carries the corresponding amino acid on its 3’ end. The enzyme aminoacyl-tRNA synthetase charges each tRNA with the correct amino acid.

Gene Regulation

Gene expression is regulated to ensure that genes are turned on or off as needed.

Genes can be:

• Constitutive: always expressed.

• Inducible: expressed only under certain conditions.

Regulation can occur at many levels, but transcription initiation is the most common control point.

In positive regulation, activator proteins enhance transcription by helping RNA polymerase bind to the promoter. In negative regulation, repressor proteins bind to operator sequences, blocking transcription.

The lac operon is a classic example of transcriptional regulation in prokaryotes. It encodes enzymes needed to metabolize lactose:

• In the absence of lactose, the LacI repressor binds the operator, blocking transcription.

• When lactose is present, it binds LacI, causing it to release the operator.

• When glucose is low, cAMP levels rise, and CAP (catabolite activator protein) binds near the promoter to enhance transcription.

Thus, the operon is most active when lactose is present and glucose is absent — a system called dual control. This mechanism ensures that the cell utilizes the most energetically favorable sugar source available, optimizing metabolic efficiency and resource allocation.