From Gene to Polypeptide

• Codons: The genetic instructions for a polypeptide are written in DNA as a series of non-overlapping, three-nucleotide words called codons. Each codon in mRNA specifies one of the 20 amino acids.

• Redundancy and Lack of Ambiguity: The code is redundant (multiple codons can code for the same amino acid) but not ambiguous (no single codon specifies more than one amino acid). This provides a buffer against certain mutations.

• Reading Frame: For the correct polypeptide to be produced, the cell's protein-synthesizing machinery must read the nucleotides in the correct groupings. A shift in the reading frame (e.g., by adding or deleting a base) completely alters the resulting protein.

• Universality: The genetic code is nearly universal across all known organisms, from bacteria to humans, which allows for powerful applications in biotechnology like gene splicing.

1. Initiation

• Promoter Recognition: RNA polymerase binds to a specific DNA sequence called the promoter, which signals the start of a gene. In eukaryotes, transcription factors help mediate this binding.
• Unwinding: The enzyme unwinds the DNA strands to expose the template strand.

2. Elongation

• RNA Synthesis: RNA polymerase moves along the DNA template strand in the $3' \rightarrow 5'$ direction, assembling RNA nucleotides in the $5' \rightarrow 3'$ direction.
• Base Pairing: RNA polymerase follows base-pairing rules, substituting Uracil (U) for Thymine (T) when pairing with Adenine (A).

3. Termination

• Release: Transcription continues until the polymerase reaches a termination sequence. In eukaryotes, the pre-mRNA is released for further processing, while in prokaryotes, translation can often begin immediately.

• 5' Capping and Poly-A Tail: A modified guanine cap is added to the $5'$ end, and a string of adenine nucleotides (poly-A tail) is added to the $3'$ end. These protect the mRNA from degradation and help ribosomes attach.

• RNA Splicing: Large portions of the RNA molecule are removed. The non-coding segments are called introns, while the segments that eventually get expressed are called exons.

• Spliceosomes: These complex structures of proteins and small RNAs catalyze the splicing process, joining exons together to create a continuous coding sequence.

Prokaryotes vs. Eukaryotes

• Coupling: In prokaryotes, transcription and translation occur simultaneously in the cytoplasm because there is no nuclear envelope.
• Processing: Eukaryotic mRNA must undergo significant processing (splicing, capping) before leaving the nucleus, whereas prokaryotic mRNA is typically ready for translation immediately.

• Directionality Check: Always remember that RNA polymerase reads the template DNA $3' \rightarrow 5'$ but synthesizes the new RNA strand $5' \rightarrow 3'$. If an exam gives you a DNA sequence, identify which strand is the template.

• The 'U' Rule: A common mistake is writing 'T' in an mRNA sequence. Always replace Thymine with Uracil when transcribing from DNA to RNA.

• Calculating Lengths: If a polypeptide has $n$ amino acids, it requires at least $3n$ nucleotides in the coding region of the mRNA (plus the stop codon).

• Start and Stop: Translation always begins at the AUG start codon (coding for Methionine) and ends at one of three stop codons (UAA, UAG, UGA) which do not code for amino acids.