Chapter 7: The Blueprint of Life, from DNA to Protein

A Glimpse of History

• Tsuneo Okazaki

  • Born in 1933, first generation of women in Japan to earn a university education.

  • Degree from Nagoya University in 1956.

  • Married Reiji Okazaki, legal research collaborator.

  • In 1960, traveled to the US to research DNA with Arthur Kornberg (discovered DNA polymerase).

  • Published a paper in 1968 explaining DNA replication via short DNA pieces now known as Okazaki fragments.

DNA: Blueprint of Life

• Diversity in Life:

  • Governed by information in DNA.

  • Composed of nucleotides, containing nucleobases:

  • Adenine (A)

  • Thymine (T)

  • Cytosine (C)

  • Guanine (G)

  • 3 nucleotides encode specific amino acids.

  • Amino acids form proteins.

  • The sequence determines protein structure and function (e.g., structural proteins, enzymes).

Overview of Genetic Information

Genome and Genes

• Genome: Complete set of genetic information including chromosomes and plasmids.

• All cells have a DNA genome, while some viruses may have an RNA genome.

• Gene: Function unit encoding a gene product, usually a protein; studied under genomics.

Multiplication of Cells

• Two essential tasks:

  • DNA replication

  • Gene expression:

  • Transcription - Converts DNA information into RNA.

  • Translation - Synthesizes proteins from RNA information.

Central Dogma of Molecular Biology

• Information flow:

  • DNA → RNA → Protein (illustrated in Figure 7.1).

Characteristics of DNA

Structure and Bonding

1. Double-Stranded Helix:

   • Carbon atoms of pentose sugar are numbered.

   • Nucleotides form a sugar-phosphate backbone.

   • Orientation: each strand has a 5ʹ and 3ʹ end.

2. Base Pairing:

   • Strands are complementary, linked by hydrogen bonds.

     • Adenine (A) with Thymine (T) via 2 hydrogen bonds.

     • Cytosine (C) with Guanine (G) via 3 hydrogen bonds.

   • Strands are anti-parallel.

Characteristics of RNA

• Contains ribose instead of deoxyribose.

• Uses uracil (U) in place of thymine (T).

• Generally shorter and single-stranded, synthesized from the DNA template strand.

• Base-pairing rules apply; uracil pairs with adenine (A).

Three Types of RNA

• Messenger RNA (mRNA) - carries genetic information.

• Ribosomal RNA (rRNA) - component of ribosomes.

• Transfer RNA (tRNA) - transfers amino acids during protein synthesis.

Regulating Gene Expression

• Cells control gene expression through:

  • Rapid mRNA degradation (illustrated in Figure 7.4).

DNA Replication

1. Replication Process Overview:

   • Bidirectional replication begins at the origin of replication and two forks meet at the terminating site (Figure 7.5).

2. Semiconservative Replication:

   • Each new DNA molecule contains one original strand and one newly synthesized strand (Figure 7.5).

3. Initiation of Replication:

   • Gyrase and helicases unwind DNA helix at the origin, exposing templates.

   • Primases synthesize short RNA primers.

   • Enzyme assembly lines formed called replisomes.

4. DNA Polymerases:

   • Synthesize DNA in a 5ʹ to 3ʹ direction by adding nucleotides to the 3ʹ end of the new strand.

   • Cannot initiate synthesis without a primer.

5. Leading and Lagging Strand Synthesis:

   • Leading strand synthesized continuously.

   • Lagging strand synthesized in short Okazaki fragments, followed by DNA ligase linking fragments (Figure 7.6-7.7).

6. E. coli Replication Time:

   • Takes about 40 minutes for chromosome replication; cell's optimal generation time is approximately 20 minutes.

   • Cells can initiate replication before the previous round is complete, leading to concurrent replication processes.

Gene Expression in Bacteria

1. Transcription Process:

   • RNA polymerase synthesizes RNA using DNA as a template, binding at a promoter and moving in the 5ʹ to 3ʹ direction.

   • Transcription terminates at a terminator sequence (Figure 7.8).

2. Prokaryotic mRNA Transcripts:

   • Monocistronic (single gene) or polycistronic (multiple genes) with related functions (Figure 7.10).

3. Initiation of RNA Synthesis:

   • Sigma (σ) factors guide RNA polymerase to recognize specific promoters.

Transcription Steps

• Initiation - RNA polymerase binds and melts a short stretch of DNA.

• Elongation - RNA polymerase synthesizes RNA adding nucleotides in the 5ʹ to 3ʹ direction, separating RNA from the DNA template.

• Termination - Encountering a terminator causes RNA polymerase to dissociate (Figures 7.9-7.10).

Characteristics of the Transcription Process

• Nucleotide Sequences:

  • RNA is complementary and antiparallel to the DNA template strand (Figures 7.9-7.10).

• The promoter orientation defines transcription direction and template strand used (Figure 7.11).

Components of Transcription in Bacteria

• (−) strand of DNA: Template for RNA synthesis, complementary to the RNA molecule.

• (+) strand of DNA: Complementary to the template, RNA sequence matches this strand except for uracil (U) replacing thymine (T).

• Promoter: Sequence for RNA polymerase binding.

• RNA polymerase: Enzyme synthesizing RNA; operates 5ʹ to 3ʹ.

• Sigma (σ) factor: Recognizes promoters allowing transcription control.

• Terminator: Sequence where transcription stops (Table 7.2).

Translation Overview

1. Protein Synthesis Process:

   • Decodes information in mRNA (requires mRNA, ribosomes, tRNAs).

   • The genetic code: three nucleotides (codon) represent an amino acid (Figure 7.12).

2. Genetic Code Attributes:

   • 64 codons represent 20 amino acids; 3 are stop codons signaling translation termination.

   • Genetic code is considered degenerate (more than one codon per amino acid) (Figures 7.3-7.4).

3. Transfer RNA Functionality:

   • Delivers correct amino acids; each tRNA has a specific anticodon for protein synthesis (Figure 7.13).

4. Role of Ribosomes:

   • Aligns and forms peptide bonds between amino acids; detection of start codon AUG begins translation (Figure 7.33).

5. Translation Mechanics:

   • Initiation at ribosome-binding site, elongation occurs in several steps as ribosome moves along mRNA (Figures 7.15-7.17), termination at stop codons.

Eukaryotic vs. Prokaryotic Gene Expression Differences

• Eukaryotic mRNA is complex and processing includes capping and polyadenylation, unlike simpler prokaryotic messengers (Figures 7.19-7.22).

• Introns are spliced out, leaving exons (expressed regions) in mature mRNA.

Microbial Gene Regulation

1. Sensing Environmental Changes:

   • Microbes adapt to varying environments (e.g., E. coli fluctuations in nutrient availability).

2. Signal Transduction Mechanisms:

   • Allows reaction to environmental stimuli (Figure 7.20-7.21).

3. Natural Selection Role in Gene Expression:

   • Random expression changes may enhance survival (e.g., Neisseria gonorrhoeae phase variation to evade immune responses).

4. Operon Structure:

   • Regulated genes transcribed as operons for efficient expression (e.g., lac operon) (Figure 7.22).

5. Regulatory Mechanisms:

   • Inducible genes: not routinely transcribed but activated when needed.

   • Repressible genes: regularly transcribed but can be turned off (Figures 7.23-7.24).

6. Transcriptional Regulation by Repressors:

   • Mechanisms governing transcription are critical for efficient use of resources in various conditions.

Lac Operon Case Study

1. Functionality:

   • Encodes proteins for lactose transport and degradation; expression is glucose-dependent (Figures 7.25-7.27).

2. Involvement of cAMP and CAP:

   • Low glucose levels lead to cAMP production facilitating lac operon transcription alongside lactose presence (Figure 7.27).

RNA Interference (RNAi)

• Mechanism for silencing gene expression, where short RNA strands bind the complex to mRNA for degradation (Figure 7.28).

Genomics and Bioinformatics

1. 7.1. First microbial genome published was Haemophilus influenzae in 1995.

2. Interpretation Challenges:

   • Complexity surrounding sequences, orientation, and reading frames for analysis (Table 7.3).

3. Future of Metagenomics:

   • Enables analysis of total microbial genomes within environments, expanding our understanding of microbial life and biodiversity.

4. Applications:

   • Promising discoveries in microbial life leading to novel compounds like antibiotics.