SBI4U - U3 (detailed) Replication, Transcription, Translation

Replication

DNA replication is a semi-conservative process, meaning each new DNA molecule contains one parental strand and one newly synthesized strand. The process begins with initiation at specific origins of replication (Ori) where the DNA double helix unwinds, forming replication "bubbles." In eukaryotes, there are multiple Ori sites along the chromosome, while prokaryotes, with smaller circular chromosomes, typically have a single Ori. The unwinding is facilitated by helicase, which breaks the hydrogen bonds between base pairs, and topoisomerase which relieves strain by cutting and rejoining the DNA ahead of the replication fork. Single-stranded binding proteins stabilize the separated strands, preventing them from re-annealing. To start synthesis, primase builds a short RNA primer at each Ori, providing a 3' hydroxyl group necessary for DNA polymerase III to add nucleotides.

Elongation occurs when DNA polymerase III synthesizes the new strands by adding deoxynucleoside triphosphates (dNTPs) to the growing DNA chain in the 5' to 3' direction, using the parental strand as a template. On the leading strand, synthesis is continuous as it follows the opening of the replication fork, whereas on the lagging strand, synthesis is discontinuous due to its antiparallel orientation. The lagging strand forms in Okazaki fragments, each initiated by a new RNA primer. DNA polymerase I later replaces these RNA primers with DNA, and DNA ligase seals the gaps between fragments by forming phosphodiester bonds.

Termination of replication occurs when the entire DNA molecule has been copied. In eukaryotes, the linear chromosomes pose a challenge at their ends, as DNA polymerase cannot fully replicate the terminal regions, leaving a single-stranded overhang. This gets recognized as an error and removed by repair enzymes, causing gradual shortening of chromosomes with each replication cycle. Eukaryotic cells use telomeres, repetitive DNA sequences at the chromosome ends, to protect genes from erosion. However, telomeres shorten progressively with each cell division, eventually leading to cell senescence or death. In certain cells, such as stem cells, the enzyme telomerase extends telomeres, enabling continued replication without loss of essential DNA.

Transcription

Transcription is the process by which a gene's DNA sequence is copied into a complementary RNA sequence by RNA polymerase.

Initiation begins when RNA polymerase and other transcription factors bind to the promoter region of the gene, located slightly upstream from the coding sequence. This promoter often includes a TATA box, a stretch rich in T and A bases, which facilitates strand separation due to their weaker double hydrogen bonds. Although the promoter itself is not transcribed, it directs RNA polymerase to the correct starting site, strand, and direction.

During elongation, RNA polymerase moves along the DNA, unwinding a small transcription bubble of 10-20 base pairs, synthesizing RNA in the 5' to 3' direction. Unlike DNA replication, RNA polymerase does not require a primer to begin and uses free ribonucleotide triphosphates (rNTPs). As RNA polymerase advances, the DNA rewinds behind it. Only the template strand (running 3’to 5’) is copied to produce a complementary RNA strand (running 5’ to 3’). The non-template strand has the same sequence as the mRNA, except that thymine (T) is replaced with uracil (U).

Termination occurs when RNA polymerase reaches a termination sequence, causing it to detach from the DNA and release a pre-mRNA molecule containing both introns and exons.

Eukaryotic cells modify the pre-mRNA to produce functional, mature mRNA. A key step in this processing is RNA splicing, during which non-coding introns are removed and coding exons are joined together. This process is performed by the spliceosome, a large complex made up of proteins and small nuclear RNAs (snRNAs). The spliceosome recognizes specific sequences at the intron-exon boundaries, removes the introns, and links the exons to form a continuous coding sequence. The presence of introns allows for alternative splicing, enabling a single pre-mRNA to be spliced in different ways, generating diverse mRNA variants and, consequently, different proteins from the same gene, increasing protein diversity. In addition to splicing, other modifications include the addition of a 5' cap, a modified guanine nucleotide, and a poly-A tail, a series of adenine nucleotides added to the 3' end, which protects the mRNA from degradation, facilitates ribosome binding, and assists in nuclear export. During splicing, thee removed introns form a looped structure known as an intron lariat, which is later degraded by enzymes, recycling its components. After all modifications, the processed mRNA, now called mature mRNA, contains only exons and is ready to exit the nucleus and direct protein synthesis in the cytoplasm through translation.

Translation

Translation is the process of converting mRNA into a polypeptide chain, the primary structure of a protein. It begins with initiation; mRNA is read 5’ to 3’ so the initiation complex forms around the 5’ end. The small ribosomal subunit binds to the mRNA’s 5’ cap and UTR, and an initiator tRNA carrying methionine pairs with the start codon (AUG) to establish the reading frame. The large ribosomal subunit then attaches, and the Met-tRNA is placed in the P site. Initiation factors, using energy from GTP hydrolysis, help assemble the complex.

In elongation, the ribosome, made up of rRNA and proteins reads the mRNA in codons, each coding for an amino acid. The process follows four steps: (1) codon recognition, where a tRNA with the correct amino acid binds to the Aminoacyl tRNA (A )site; (2) peptide bond formation, where rRNA catalyzes a peptide bond between amino acids while releasing the tRNA from the A site and its amino acid; (3) translocation, where the mRNA shifts, moving the tRNA from the A to the P site; and (4) exit, where the empty tRNA leaves through the E site. Elongation factors bring the activated tRNAs to the A site, and the mRNA moves through the ribosome, building the polypeptide chain.

Finally, termination occurs when the ribosome reaches a stop codon (UAA, UAG, or UGA), triggering the release factor to add a water molecule instead of an amino acid. This causes the ribosome to dissociate, releasing the completed polypeptide chain, which may require folding and post-translational modifications to become functional.

• The newly synthesized polypeptide chain is a linear sequence of amino acids linked by peptide bonds.

• This sequence is the primary structure of the protein, and it determines how the protein will fold into its final 3D shape.

• The polypeptide may undergo post-translational modifications, such as:

  • Folding: The chain folds into complex structures (secondary, tertiary, and quaternary structures) stabilized by various bonds and interactions.

  • Chemical Modifications: Adding phosphate, methyl, or acetyl groups to amino acids to regulate the protein’s function.

  • Cleavage: Some polypeptides are cut into smaller segments, each of which may have different functions or activation states.

• The polypeptide folds into a specific three dimensional structure, forming the mature protein.

• The final shape (tertiary or quaternary structure) is essential for the protein’s function, allowing it to interact with other molecules and perform its biological role.

• Protein folding can be assisted by chaperone proteins, which prevent misfolding and ensure the protein reaches its proper shape.

• Once folded, the mature protein may:

  • Act as an enzyme to catalyze biochemical reactions.

  • Serve as a structural component of cells and tissues.

  • Function in cell signaling, immune response, transport, or other cellular processes.

  • Proteins are essential for virtually every cellular function, embodying the "output" of the central dogma, where DNA ultimately codes for functional molecules in the cell.