LEC 13Nucleic Acid Synthesis: Replication and Transcription

Nucleic Acid Synthesis: Replication and Transcription

Central Dogma of Molecular Biology

The central dogma describes the flow of genetic information in cells: DNA is replicated, transcribed into RNA, and translated into protein. Each step is essential for the storage, transfer, and expression of genetic information.

• Replication: DNA is copied to produce identical DNA molecules.

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

• Translation: RNA is used to synthesize proteins.

• Each process has three phases: Initiation, Elongation, and Termination.

Structure and Function of Nucleic Acids

Nucleic acids (DNA and RNA) are polymers of nucleotides. Their structure enables the storage and transfer of genetic information through complementary base pairing.

• DNA: Double-stranded helix, bases are adenine (A), guanine (G), cytosine (C), and thymine (T).

• RNA: Usually single-stranded, bases are A, G, C, and uracil (U).

• Complementarity is key: A pairs with T (or U in RNA), G pairs with C.

Energy of Nucleic Acid Synthesis

Phosphoanhydride Hydrolysis

The synthesis of nucleic acids is driven by the hydrolysis of phosphoanhydride bonds in nucleoside triphosphates (NTPs for RNA, dNTPs for DNA). This reaction is highly exergonic and provides the energy required for polymerization.

• General reaction: NTP + H2O → NDP + Pi

• For DNA: dNTP + H2O → dNDP + Pi

• Hydrolysis of ATP to ADP releases 1 ATP equivalent; ATP to AMP + PPi releases 2 ATP equivalents.

• Pyrophosphate (PPi) hydrolysis is considered irreversible, driving the reaction forward.

Key Equations:

• $\mathrm{ATP} + \mathrm{H}_2\mathrm{O} \rightarrow \mathrm{ADP} + \mathrm{P}_i$

• $\mathrm{ATP} + \mathrm{H}_2\mathrm{O} \rightarrow \mathrm{AMP} + \mathrm{PP}_i$

• $\mathrm{PP}_i + \mathrm{H}_2\mathrm{O} \rightarrow 2\mathrm{P}_i$

DNA Replication

DNA Polymerase Reaction

DNA polymerase catalyzes the addition of nucleotides to the growing DNA strand during replication. The enzyme uses the energy from hydrolysis of two phosphoanhydride bonds (2 ATP equivalents) per nucleotide added.

• Reaction: DNAn-OH (3') + dNTP → DNAn+1 + PPi

• PPi + H2O → 2 Pi (irreversible step)

• DNA polymerase only adds nucleotides to the 3' OH of the primer strand.

• Template strand determines which base is added via complementarity.

Semiconservative Replication: Each new DNA molecule consists of one old strand and one new strand.

Replication Initiation

Replication begins at specific sites called origins of replication, which are rich in AT base pairs. Several proteins are required to initiate replication:

• Helicase: Unwinds the DNA double helix using ATP hydrolysis.

• Topoisomerase: Relieves strain from supercoiled DNA ahead of the fork.

• Single-stranded binding proteins: Stabilize unwound DNA.

• Primase: Synthesizes a short RNA primer for DNA polymerase to extend.

Replication Elongation and Termination

DNA polymerase synthesizes new DNA in the 5'→3' direction. The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously in Okazaki fragments.

• DNA clamp: Increases processivity, allowing polymerase to add thousands of nucleotides before dissociating.

• Proofreading: DNA polymerase has 3'→5' exonuclease activity to correct errors.

• Termination: RNA primers are removed by endonucleases, and DNA ligase joins Okazaki fragments using ATP hydrolysis.

• Telomeres: Repetitive DNA sequences at chromosome ends, extended by telomerase to allow complete replication.

Transcription: DNA to RNA

Types of RNA

Three main types of RNA are involved in the central dogma:

• Messenger RNA (mRNA): Carries genetic information from DNA to ribosome.

• Transfer RNA (tRNA): Brings amino acids to the ribosome during translation.

• Ribosomal RNA (rRNA): Forms the core of the ribosome and catalyzes protein synthesis.

Transcription Initiation and Gene Structure

Transcription begins at promoter regions, often rich in AT base pairs (e.g., TATA box). Transcription factors bind promoters and recruit helicase to unwind DNA. The gene structure includes:

• 5' untranslated region (5' UTR): Contains promoter and regulatory sequences.

• Coding region: Sequence that is transcribed and translated.

• 3' untranslated region (3' UTR): Involved in regulation and termination.

RNA Polymerase Reaction

RNA polymerase synthesizes RNA from a DNA template, using the energy from hydrolysis of two phosphoanhydride bonds per nucleotide added. The mRNA sequence is complementary to the template (antisense) strand and matches the coding (sense) strand, except U replaces T.

• Reaction: RNAn-OH (3') + NTP → RNAn+1 + PPi

• PPi + H2O → 2 Pi

Transcription Elongation

Phosphorylation of RNA polymerase allows elongation to begin. RNA polymerase is processive and has proofreading ability, though less accurate than DNA polymerase. Multiple RNA polymerases can transcribe the same gene simultaneously.

Transcription Termination and mRNA Processing

Transcription termination occurs via RNA-dependent or protein-dependent mechanisms. Eukaryotic mRNAs are processed after transcription:

• 5' and 3' end modifications: Cap and poly-A tail added.

• Splicing: Introns are removed, exons are retained and translated.

• Only mature, spliced mRNA is translated.

Summary Table: Key Proteins and Functions in Replication

Protein	Function
Helicase	Unwinds DNA double helix
Topoisomerase	Relieves supercoiling strain
Single-stranded binding protein	Stabilizes unwound DNA
Primase	Synthesizes RNA primer
DNA polymerase	Synthesizes new DNA, proofreads
DNA clamp	Increases processivity
DNA ligase	Joins Okazaki fragments
Telomerase	Extends telomeres

Summary Table: Types of RNA

Type	Function
mRNA	Template for protein synthesis
tRNA	Transfers amino acids to ribosome
rRNA	Forms ribosome, catalyzes translation