Gene Expression: From Gene to Protein

Central Dogma of Molecular Biology

The central dogma of molecular biology describes the unidirectional flow of genetic information within a biological system. It explains how genetic information stored in DNA is used to synthesize proteins, which are essential for cellular function.

• Transcription: The process by which RNA is synthesized using DNA as a template.

• Translation: The process by which proteins are synthesized using the encoded messages of mRNA.

• Gene Expression: Sometimes, transcription and translation are collectively referred to as gene expression, which is the process by which genotype becomes expressed as phenotype.

• Irreversibility: While DNA can be replicated and RNA can be reverse-transcribed into DNA, the transfer of information from nucleic acid to protein is irreversible.

Introduction to Transcription

Transcription is the process that builds RNA using DNA within a gene as the coding template. Genes are small units of DNA that encode a product, such as a protein or RNA. Specific DNA sequences mark where transcription begins and ends:

• Promoter: DNA sequence where transcription starts (site of RNA polymerase attachment).

• Terminator: DNA sequence where transcription ends.

• RNA Polymerase: Enzyme that synthesizes RNA from scratch, without the need for a primer.

• Upstream/Downstream: 'Upstream' refers to DNA sequences in the opposite direction of transcription; 'downstream' refers to sequences in the direction of transcription.

Overview of Transcription

The two strands of DNA in a gene are referred to as the coding strand and the template strand. The RNA molecule produced has the same sequence as the coding strand (except uracil replaces thymine) and is synthesized from the 5' to 3' end by pairing free RNA nucleotides on the DNA template strand.

• Base Pairing: Watson & Crick base-pairing rules apply: A pairs with U (in RNA), and C pairs with G.

• Directionality: RNA is synthesized in the 5' to 3' direction.

Steps of Transcription

Transcription consists of three main steps:

1. Initiation: RNA polymerase binds to the promoter and unwinds the DNA strands. In prokaryotes, RNA polymerase binds directly; in eukaryotes, transcription factors are required.

2. Elongation: RNA polymerase synthesizes the RNA molecule by pairing free RNA nucleotides with the DNA template, moving along the DNA and unwinding it as it goes.

3. Termination: Transcription ends when RNA polymerase reaches the terminator sequence. In eukaryotes, this produces a pre-mRNA that requires further processing.

Eukaryotic RNA Processing & Splicing

Unlike prokaryotic mRNA, eukaryotic mRNA requires further modification after transcription. The initial transcript is called pre-mRNA, which undergoes RNA processing and splicing to become mature mRNA ready for translation.

• RNA Processing: Involves the addition of a 5' cap (modified guanine nucleotide) and a poly-A tail (sequence of adenine nucleotides) to the 3' end. These modifications facilitate export from the nucleus, protect mRNA from degradation, and help ribosomes attach for translation.

• RNA Splicing: Removes noncoding regions (introns) from pre-mRNA and joins coding regions (exons). The spliceosome, a complex of RNA and protein, is responsible for this process. Alternative splicing allows a single gene to code for multiple proteins.

Types of RNA

Cells use several types of RNA, each with distinct functions:

• Messenger RNA (mRNA): Carries genetic information from DNA and is translated into protein. Contains codons (three-nucleotide sequences coding for specific amino acids).

• Ribosomal RNA (rRNA): Forms part of the structure of ribosomes.

• Transfer RNA (tRNA): Carries amino acids to the ribosome during translation. Contains anticodons complementary to mRNA codons.

The Genetic Code

The genetic code is a set of rules by which information encoded in DNA or RNA sequences is translated into proteins by living cells. It is nearly universal and is read in triplets called codons, each specifying a particular amino acid.

• Redundancy: The code is redundant, meaning multiple codons can code for the same amino acid.

• Start Codon: AUG codes for methionine and signals the start of translation.

• Stop Codons: UAA, UAG, and UGA signal the end of translation.

Introduction to Translation

Translation is the process by which proteins are synthesized from mRNA templates. Ribosomes facilitate the decoding of mRNA into a polypeptide chain, with tRNA molecules bringing the appropriate amino acids.

• Ribosomes: Composed of large and small subunits made of rRNA and proteins. Prokaryotic ribosomes are 70S (50S + 30S), and eukaryotic ribosomes are 80S (60S + 40S).

• tRNA: Each tRNA has an anticodon that pairs with the mRNA codon and an amino acid attachment site.

• Charged tRNA: tRNA attached to an amino acid; discharged tRNA is not attached to an amino acid.

Steps of Translation

Translation occurs in three main steps:

1. Initiation: The small ribosomal subunit binds to mRNA and a tRNA carrying methionine (AUG start codon), followed by the large subunit. Initiation factors and energy are required.

2. Elongation: Amino acids are added one by one to the growing polypeptide chain. The ribosome moves along the mRNA in the 5' to 3' direction, and tRNAs enter the A site, move to the P site, and exit via the E site.

3. Termination: When a stop codon is reached, release factors bind, causing the polypeptide to be released and the translation complex to dissociate.

Post-Translational Modification

After translation, proteins often undergo post-translational modifications (PTMs) that regulate their activity, localization, and stability. Common PTMs include methylation, acetylation, ubiquitination, phosphorylation, and glycosylation.

• Glycosylation: Addition of carbohydrates to proteins.

• Phosphorylation: Addition of phosphate groups, which can activate or deactivate proteins.

Review: Transcription vs. Translation

The following table summarizes the key differences between transcription and translation:

Mutations

Mutations are permanent changes in the DNA sequence. They can affect gene expression and protein function, and may be harmful, beneficial, or neutral. Mutations can occur spontaneously or be induced by environmental factors (mutagens). Types of mutations include:

• Substitution: One base is replaced by another.

• Insertion: Addition of one or more nucleotides.

• Deletion: Removal of one or more nucleotides.

• Frameshift: Insertions or deletions that alter the reading frame of the gene.

• Missense: Substitution that changes one amino acid.

• Nonsense: Substitution that creates a stop codon, terminating translation prematurely.

• Silent: Substitution that does not change the amino acid sequence due to redundancy in the genetic code.

Additional info: Mutations are a primary source of genetic diversity and evolution in populations.