Gene Expression: From Gene to Protein (Transcription and RNA Processing)

Gene Expression: From Gene to Protein

Introduction to Gene Expression

Gene expression is the process by which information encoded in DNA directs the synthesis of proteins, ultimately determining the phenotype of an organism. This process involves two main stages: transcription and translation.

• All cells contain the same DNA, but different cell types (e.g., skin, brain, liver) express different sets of genes, leading to cellular diversity.

• Gene expression is regulated by turning specific genes on or off in different cells.

Overview: The Flow of Genetic Information

• The information content of genes is stored as specific sequences of nucleotides in DNA.

• DNA dictates the synthesis of proteins and RNA molecules involved in protein synthesis.

• Proteins are the link between genotype (genetic makeup) and phenotype (observable traits).

• Gene expression includes two main stages: transcription (DNA to RNA) and translation (RNA to protein).

Central Dogma of Molecular Biology

• Replication: DNA is copied to produce more DNA.

• Transcription: DNA is used as a template to synthesize RNA.

• Translation: RNA is used as a template to synthesize proteins.

Reverse transcription (in some viruses) allows RNA to be copied back into DNA.

Some RNA molecules function without being translated into protein (e.g., rRNA, tRNA).

Prions are infectious proteins that can propagate without nucleic acids.

Nutritional Mutants: Scientific Inquiry

Studies with Neurospora mutants helped elucidate the relationship between genes and enzymes.

• Mutants requiring arginine for growth were found to be blocked at different steps in the arginine biosynthesis pathway.

Gene	Enzyme	Step in Pathway
Gene A	Enzyme A	Precursor → Ornithine
Gene B	Enzyme B	Ornithine → Citrulline
Gene C	Enzyme C	Citrulline → Arginine

Conclusion: Each gene encodes a specific enzyme (or polypeptide) in a metabolic pathway.

The Products of Gene Expression: A Developing Story

• Not all proteins are enzymes; the "one gene-one enzyme" hypothesis was revised to "one gene-one protein" and then to "one gene-one polypeptide" hypothesis.

• Many proteins are composed of multiple polypeptides, each encoded by a separate gene.

• Gene products are commonly referred to as proteins.

Basic Principles of Transcription and Translation

RNA as the Bridge

• RNA is chemically similar to DNA but contains ribose sugar and uracil (U) instead of thymine (T).

• RNA is usually single-stranded.

• Transcription produces messenger RNA (mRNA), which carries genetic information from DNA to the ribosome.

• Translation synthesizes a polypeptide using the information in mRNA.

Transcription and Translation in Prokaryotes vs. Eukaryotes

• In bacteria, translation can begin before transcription is finished.

• In eukaryotes, the nuclear envelope separates transcription (in the nucleus) from translation (in the cytoplasm).

• Eukaryotic RNA transcripts are modified through RNA processing to yield mature mRNA.

• A primary transcript is the initial RNA transcript from any gene prior to processing.

The Genetic Code

• There are only four nucleotide bases (A, T, C, G) to specify 20 amino acids.

• The genetic code is based on a triplet code: three-nucleotide sequences (codons) specify amino acids.

Codons: Triplets of Nucleotides

• The flow of information from gene to protein is based on nonoverlapping, three-nucleotide words (codons).

• During transcription, one DNA strand (the template strand) provides the template for ordering the sequence of complementary nucleotides in an RNA transcript.

• During translation, mRNA codons are read in the 5' to 3' direction, each specifying an amino acid.

• The nontemplate DNA strand is called the coding strand.

Cracking the Code

• All 64 codons were deciphered by the mid-1960s.

• Of the 64 codons, 61 code for amino acids; 3 are stop signals.

• The genetic code is redundant (more than one codon may specify an amino acid) but not ambiguous (no codon specifies more than one amino acid).

• Codons must be read in the correct reading frame for the correct polypeptide to be produced.

Reading Frame Example

• 5' ATG CTA AG 3' can be read as ATG-CTA-AG, A-TGC-TAA-G, or AT-GCT-AAG, depending on the starting point.

Evolution of the Genetic Code

• The genetic code is nearly universal, shared by all living organisms.

• Genes can be transcribed and translated after being transplanted from one species to another.

• This universality suggests a common evolutionary origin.

Transcription: DNA-Directed Synthesis of RNA

Why Have RNA as an Intermediate?

• RNA acts as an intermediary, allowing the genetic information in DNA to be used for protein synthesis without altering the DNA itself.

Molecular Components of Transcription

• RNA polymerase is the enzyme that synthesizes RNA by joining together complementary RNA nucleotides using the DNA template.

• RNA polymerase assembles polynucleotides in the 5' to 3' direction and does not require a primer.

Stages of Transcription

• Initiation: RNA polymerase binds to the promoter, DNA unwinds, and RNA synthesis begins.

• Elongation: RNA polymerase moves along the DNA, untwisting the double helix and elongating the RNA strand.

• Termination: RNA synthesis ends, and the RNA transcript is released.

Promoters, Terminators, and Transcription Factors

• The promoter is a DNA sequence where RNA polymerase attaches to initiate transcription.

• The terminator signals the end of transcription (in bacteria).

• A transcription unit is the stretch of DNA that is transcribed.

• Transcription factors are proteins that help RNA polymerase bind to the promoter in eukaryotes, forming the transcription initiation complex.

Elongation and Termination in Detail

• As RNA polymerase moves, it untwists the DNA helix and synthesizes RNA at about 40 nucleotides per second in eukaryotes.

• Multiple RNA polymerases can transcribe a gene simultaneously.

• Termination differs between bacteria and eukaryotes:

  • In bacteria, transcription ends at the terminator, and mRNA is ready for translation.

  • In eukaryotes, RNA polymerase II transcribes a polyadenylation signal sequence; the transcript is released 10–35 nucleotides past this sequence.

Eukaryotic Cells Modify RNA After Transcription

RNA Processing

• Enzymes in the nucleus modify pre-mRNA before it is exported to the cytoplasm.

• Both ends of the primary transcript are altered:

  • A 5' cap (modified guanine nucleotide) is added to the 5' end.

  • A poly-A tail (50–250 adenine nucleotides) is added to the 3' end.

• These modifications protect mRNA from degradation and help with export and translation.

Split Genes and RNA Splicing

• Many eukaryotic genes contain introns (noncoding regions) and exons (coding regions).

• During RNA splicing, introns are removed, and exons are joined together to form mature mRNA.

• Alternative RNA splicing allows a single gene to code for multiple polypeptides, depending on which exons are included.

• RNA splicing is carried out by spliceosomes, complexes of proteins and small RNAs.

Summary Table: Key Steps in Eukaryotic Gene Expression

Step	Location	Main Events
Transcription	Nucleus	DNA → pre-mRNA
RNA Processing	Nucleus	5' cap, poly-A tail, splicing
Translation	Cytoplasm	mRNA → Protein

Example: The human beta-globin gene contains three exons and two introns. Alternative splicing can produce different forms of beta-globin protein.

Additional info: The universality of the genetic code is a foundation for genetic engineering, allowing genes from one organism to be expressed in another (e.g., production of human insulin in bacteria).