Gene Expression: From Gene to Protein

Basic Principles of Transcription and Translation

Gene expression is the process by which genetic information encoded in DNA directs the synthesis of proteins, the molecules responsible for most cellular functions. This process involves two main stages: transcription and translation, with RNA serving as the intermediary between DNA and protein synthesis.

• Genes contain the instructions for building proteins.

• RNA acts as the bridge between DNA and protein synthesis.

  • RNA is chemically similar to DNA but contains ribose sugar instead of deoxyribose.

  • RNA uses uracil (U) instead of thymine (T).

  • RNA is usually single-stranded.

• Transcription: The synthesis of RNA from a DNA template, producing messenger RNA (mRNA).

• Translation: The synthesis of a polypeptide (protein) from the mRNA transcript, occurring at ribosomes.

• Bacteria: Translation can begin before transcription ends due to the absence of a nucleus.

• Eukaryotes: The nuclear envelope separates transcription (in the nucleus) from translation (in the cytoplasm). RNA transcripts undergo processing before becoming mature mRNA.

The Genetic Code

The genetic code specifies which amino acids will be used to build a protein. It is universal, redundant, and unambiguous.

• Redundant: More than one codon can specify a particular amino acid.

• Not ambiguous: Each codon specifies only one amino acid.

Codons: Triplets of Nucleotides

• Codon: A sequence of three nucleotides on mRNA that specifies a particular amino acid.

• Each codon is recognized by a tRNA with a complementary anticodon.

• There are more codons (64) than amino acids (20).

• Start codon: AUG (codes for methionine; signals initiation of translation).

• Stop codons: UAA, UAG, UGA (signal termination of translation).

Transcription: DNA-Directed Synthesis of RNA

Molecular Components of Transcription

Transcription is the process by which RNA is synthesized from a DNA template. It involves several steps and key molecular players.

• RNA polymerase: The enzyme that catalyzes RNA synthesis by adding RNA nucleotides to the DNA template strand in the 5’ to 3’ direction.

• Promoter: A DNA sequence signaling the start point for transcription; RNA polymerase binds here to initiate transcription.

• RNA polymerase does not require a primer to begin synthesis.

Stages of Transcription

1. Initiation: RNA polymerase binds to the promoter and unwinds the DNA.

2. Elongation: RNA polymerase moves along the DNA, untwisting the double helix and synthesizing RNA by adding nucleotides to the 3’ end of the growing strand. In eukaryotes, this occurs at about 40 nucleotides per second.

3. Termination:

   • Bacteria: RNA polymerase stops at a terminator sequence; mRNA is ready for translation.

   • Eukaryotes: Transcription continues past the polyadenylation signal (AAUAAA); pre-mRNA is released for further processing.

Eukaryotic RNA Processing

RNA Processing

In eukaryotes, the primary RNA transcript (pre-mRNA) undergoes several modifications before it is translated into protein.

• Both ends of the RNA are modified:

  • 5’ cap: Addition of a modified guanine nucleotide to the 5’ end.

  • 3’ poly-A tail: Addition of a string of adenine nucleotides to the 3’ end.

• Functions of these modifications:

  • Facilitate export of mRNA from the nucleus.

  • Protect mRNA from degradation by enzymes.

  • Help ribosomes attach to the 5’ end for translation.

Split Genes and RNA Splicing

• Introns: Non-coding regions of RNA that are removed.

• Exons: Coding regions that are spliced together to form the mature mRNA.

Translation: RNA-Directed Synthesis of a Polypeptide

Molecular Components of Translation

Translation is the process by which the sequence of an mRNA is decoded to build a polypeptide, with the help of tRNA and ribosomes.

Transfer RNA (tRNA)

• tRNA molecules recognize and bind specific amino acids and match them to the appropriate codons in mRNA via their anticodon.

• Each tRNA is about 80 nucleotides long and folds into an L-shaped 3D structure.

• Charged tRNA: tRNA bound to its specific amino acid.

Ribosomes

• Ribosomes facilitate the coupling of tRNA anticodons with mRNA codons.

• Composed of protein and ribosomal RNA (rRNA), forming large and small subunits.

• Three binding sites:

  • A site: Holds the tRNA carrying the next amino acid to be added.

  • P site: Holds the tRNA carrying the growing polypeptide chain.

  • E site: Site from which discharged tRNAs exit the ribosome.

Stages of Translation

1. Initiation:

   • mRNA binds to the small ribosomal subunit.

   • The subunit moves along mRNA to the start codon (AUG).

   • Charged tRNA enters the P site; large subunit joins to form the complete ribosome.

2. Elongation:

   • Charged tRNA enters the A site.

   • Peptide bond forms between amino acids in the P and A sites.

   • tRNA in P site moves to E site and exits; tRNA in A site moves to P site; new tRNA enters A site.

3. Termination:

   • Occurs when a stop codon reaches the A site.

   • A release factor binds, causing the addition of a water molecule instead of an amino acid, releasing the polypeptide.

   • The translation complex disassembles.

• All stages require protein factors and energy (GTP hydrolysis).

Protein Folding and Post-Translational Modifications

• Polypeptide chains spontaneously fold into their functional 3D shapes during synthesis.

• Post-translational modifications may include addition of sugars, lipids, phosphate groups, or cleavage of the polypeptide.

• Multiple polypeptides may assemble into a functional protein.

Polyribosomes

• Multiple ribosomes can simultaneously translate a single mRNA, forming a structure called a polyribosome or polysome.

• This increases the efficiency of protein synthesis.

Mutations: Effects on Protein Structure and Function

Mutations

Mutations are changes in the genetic material of a cell. They can affect protein structure and function, sometimes causing disease.

• Point mutations: Chemical changes in a single nucleotide pair of a gene.

• Can lead to abnormal proteins; if in gametes, may be inherited (e.g., sickle-cell disease).

Types of Small-Scale Mutations

• Single nucleotide-pair substitutions

• Nucleotide-pair insertions or deletions

Substitutions

• Silent mutations: No effect on amino acid sequence due to redundancy in the genetic code.

• Missense mutations: Change one amino acid to another; may affect protein function.

• Nonsense mutations: Change an amino acid codon into a stop codon, usually resulting in a nonfunctional protein.

Insertions and Deletions

• Additions or losses of nucleotide pairs in a gene.

• Often have more severe effects than substitutions.

• May cause frameshift mutations, altering the reading frame and usually resulting in a nonfunctional protein.

Summary Table: Types of Point Mutations and Their Effects

Key Equations and Concepts

• Central Dogma of Molecular Biology:

• Transcription Direction:

• Genetic Code: 64 codons, 20 amino acids, 3 stop codons, 1 start codon (AUG).

Example: A point mutation in the hemoglobin gene (GAG to GTG) causes sickle-cell disease by substituting valine for glutamic acid.

Additional info: The universality of the genetic code allows genes from one organism to be expressed in another, forming the basis for genetic engineering and biotechnology.