Molecular Biology: Genes and Expression

Gene

• Is the fundamental unit of inheritance and determines all phenotypes.
• Every gene contains instructions for a specific protein.
• The human genome contains an estimated 30,000 to 120,000 genes.
• Only a fraction of genes are expressed in a particular cell at any given time.

Gene Structure

• Most eukaryotic genes consist of coding sequences (exons) interrupted by noncoding DNA (introns).
• The average gene has 7-10 exons spread over 10-16kb of DNA.
• Gene structure includes exons, start signals, stop signals, and regulatory control elements.
• The two main functional units are the promoter region and the coding region.

Gene Expression

• The process by which a gene's information is converted into a biologically functional molecule of either protein or RNA.
• Gene expression is assumed to be controlled at various points in the sequence leading to protein synthesis.
• The central dogma of molecular biology describes the flow of genetic information: DNA → RNA → protein.
  • Transcription: DNA → RNA
  • Translation: RNA → protein

Types of Genes

• Constitutive genes (Housekeeping genes)
  • Are expressed at a constant rate in everyday processes.
  • Not subjected to regulation.
  • Examples include enzymes of glycolysis.
• Regulated genes
  • Expressed only under certain conditions.
  • Expressed in all cells or a subset of cells.
  • Example: the expression of the insulin gene in the pancreas.

Flow of Genetic Information in Prokaryotes vs. Eukaryotes

• Prokaryotes
  • Transcription and translation occur simultaneously.
• Eukaryotes
  • Transcription occurs in the nucleus followed by RNA processing.
  • Translation occurs in the cytoplasm.

Eukaryotic Promoter

• Is a region of DNA that initiates transcription of a particular gene.
• Located near the transcription start sites of genes, upstream on the DNA (towards the 5’ region of the sense strand).
• Promoters can be about 100–1000 base pairs long.
• Conserved eukaryotic promoter elements
  • CAAT box
  • TATA box
  • GC box
  • CAP site
• Consensus sequence
  • GGCCAATCT (CAAT box)
  • TATAA (TATA box)
  • GGGCGG (GC box)
  • TAC (CAP site)

Enhancer Sequences

• Are regulatory DNA sequences that enhance the transcription of an associated gene when bound by specific proteins called transcription factors.
• Can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or thousands of nucleotides away.

Silencers

• Are regions of DNA that depress the rate of transcription when bound by specific transcription factors called repressors.

Transcription

• Is the process by which certain areas of DNA are copied (transcribed) to mRNA, which carries the information needed for protein synthesis.
• Processes include DNA replication, DNA repair, genetic recombination, and RNA synthesis (transcription).

Messenger RNA (mRNA)

• mRNA is a type of RNA that is necessary for protein production.
• It uses the information in genes to create a blueprint for making proteins.

Reverse Transcription

• Is the synthesis of DNA from an RNA template catalyzed by reverse transcriptase enzyme.
• Retroviruses contain RNA as their genetic material.
• The retroviral RNA serves as a template for synthesis of DNA by reverse transcriptase.
• Examples of retroviruses: HIV (AIDS), Hepatitis C.

Transcription in Eukaryotes

• Pre-messenger RNA is formed, with the involvement of RNA polymerase enzymes.
  • RNA Polymerase Reads DNA and makes an RNA copy of one strand/one gene.

RNA Polymerase

• RNA polymerases I and III transcribe rRNA and tRNA genes, respectively.
• Pol III transcribes a few other RNAs.
• All three are big, multimeric proteins (500-700 kD).
• All have two large subunits with sequences similar to those in E.coli RNA polymerase (Prokaryotes).
• Pol II is the most sensitive to α-amanitin, an octapeptide from Amanita phalloides ("destroying angel mushroom").

Transcription Factors

• The three polymerases (I, II and III) interact with their promoters via so-called transcription factors.
• Transcription factors recognize and initiate transcription at specific promoter sequences.

Transcription in Eukaryotes: Post-Transcriptional Modifications or Gene Processing

• The process by which primary transcript RNA is converted into mature RNA.
• The pre-mRNA molecule undergoes three main modifications:
  • 5' Capping
  • 3' polyadenylation
  • RNA splicing

Capping

• Involves the addition of 7-methylguanosine (m7G) to the 5' end of the pre-mRNA.
• Protects the 5' end of the primary RNA transcript from attack by ribonuclease.

Cleavage and Polyadenylation

• Involves cleavage of the 3' end of the pre-mRNA molecule and the addition of about 250 adenine residues to form a poly(A) tail.
• Protects the 3' end from ribonuclease digestion.

RNA Splicing 

• The pre-messenger RNA contains introns that are not required for protein synthesis.
• The sequence AAUAAA in hnRNA serves as a signal for cleavage and addition of the poly(A) tail.
• The pre-messenger RNA is chopped up to remove the introns and create messenger RNA (mRNA).

The Structure of a Eukaryotic Gene and its Products

• 5' flanking region
  • Promoter
• Transcribed region
  • Intron
  • Exon
• Enhancer
• CAAT box
• GC boxes
• TATA box
• Protein
  • Cap site
  • Start signal
• Left splice site
• Right splice site
• Protein stop signal
• Poly (A) addition signal
• Polyadenylation site

Synthesis of Messenger RNA (mRNA) in Eukaryotes: hnRNA, heterogeneous nuclear RNA.

• Nucleus
  • 5' cap
  • Intron
  • 3' poly (A)
• DNA
• Nuclear envelope
• Nuclear pore

Clinical Correlates

• The most common causes of β-thalassemia are defects in mRNA splicing of the ẞ-globin gene.
• Mutations that affect splicing create aberrant transcripts that are degraded before they are translated.
• Thalassemia minor is a mild anemia caused by inheriting a single mutated gene.
• Thalassemia major is a severe transfusion-dependent anemia caused by inheriting two mutated genes.

Synthesis of Ribosomal RNA (rRNA) and Assembly of Ribosomes

• Nuclear organizer

  • 5'
  • 3'

• Nuclear envelope

• Nucleolus

• Nuclear pore

• Cytoplasm

• 45S rRNA precursor

Synthesis of Transfer RNA (tRNA)

• Nucleus

  • Intron
  • tRNA precursor
  • tRNA
  • tRNA

• DNA

• Nuclear pore

• Cytoplasm

• tRNA modification

  • D, T, W, and (representing other modified nucleotides) are unusual nucleotides produced by post-transcriptional modifications.

Synthesis of Transfer RNA (tRNA)

• RNA polymerase III produces tRNA.
• The promoter is located within the coding region of the gene.
• Primary transcripts for tRNA are cleaved at the 5' and 3' ends.
• Some precursors contain introns that are removed.
• During processing of tRNA precursors, nucleotides are modified.
• Post-transcriptional modification includes the conversion of uridine to pseudouridine (Ψ), ribothymidine (T), and dihydrouridine (D).
• Other unusual nucleotides are also produced.
• Addition of the sequence CCA to the 3' end is catalyzed by nucleotidyl transferase.

tRNA

• Found in all tRNAs
• Not found in all tRNAs.Other variable sites are shown in blue as well
• The modified bases are:
  • I = inosine
  • ml = methylinosine
  • T = ribothymidine
  • UH₂ = dihydrouridine
  • m₂G = dimethylguanosine
  • ψ = pseudouridine

Eukaryotic Transcription

• Transcription is the process of copying DNA into RNA.
• DNA is transcribed by RNA polymerase into messenger RNA (mRNA), which carries the genetic information needed for protein synthesis.
• Eukaryotes have three RNA polymerases (I, II, and III) that are involved in transcribing different types of RNA.
• RNA polymerase II is responsible for synthesizing mRNA.

Eukaryotic Promoter

• A promoter is a DNA sequence that initiates transcription of a gene.
• Promoters are located upstream of the transcription start site of genes.
• Common eukaryotic promoter elements include:
  • CAAT box
  • TATA box
  • GC box
  • CAP site

Enhancers and Silencers

• Enhancers are regulatory DNA sequences that increase the likelihood of transcription.
• They can be located thousands of nucleotides away from the gene they enhance.
• Silencers are regulatory DNA sequences that decrease the likelihood of transcription.

Transcription Factors

• Transcription factors are proteins that bind to promoters and other regulatory sequences.
• They regulate the transcription of genes by interacting with RNA polymerase and other proteins.
• General transcription factors are required for the initiation of transcription by RNA polymerase II.
• These include: TFIID, TFIIB, TFIIF, TFIIE, and TFIIH.

Post-Transcriptional Modifications

• Pre-messenger RNA (hnRNA) is processed into mature mRNA through three main modifications:
  • 5' capping
  • 3' polyadenylation
  • RNA splicing

5' Capping

• 7-methylguanosine (m7G) is added to the 5' end of the pre-mRNA molecule.
• Capping protects the mRNA from degradation and helps initiate translation.

3' Polyadenylation

• A poly(A) tail is added to the 3' end of the pre-mRNA molecule.
• The tail protects the mRNA from degradation and helps with translation.

RNA Splicing

• Introns (non-coding sequences) are removed from the pre-mRNA.
• Exons (coding sequences) are joined together to form mature mRNA.
• Splicing is essential for producing functional mRNA.

Ribosomal RNA (rRNA) Synthesis

• rRNA is synthesized in the nucleolus, a specialized region within the nucleus.
• The 45S rRNA precursor is processed into different rRNA molecules that are assembled into ribosomes.

Transfer RNA (tRNA) Synthesis

• tRNA is synthesized in the nucleus and is responsible for transporting amino acids to the ribosome for protein synthesis.
• The primary tRNA transcript is processed into mature tRNA through cleavage, intron removal, and post-transcriptional modifications.
• Post-transcriptional modification includes the conversion of uridine to pseudouridine, ribothymidine, and dihydrouridine.
• Other modified nucleotides, like inosine and methylguanosine, are also produced.

Clinical Correlations

• Beta-thalassemia is a genetic disorder that results from defects in the splicing of the beta-globin gene.