Molecular Genetics Lesson 4

Learning Points

  • The main topics covered in this lesson are:

    • DNA replication

    • Transcription

    • Translation

DNA Replication

Models of DNA Replication

  • There are three established models of DNA replication:

    1. Conservative Model

    • Proposes that the parental molecule remains intact.

    • The resulting daughter molecule is formed from two newly synthesised DNA strands.

    • Both strands of the DNA molecule serve as templates for the synthesis of new daughter DNA.

    • The two parental strands reassociate to restore the parental molecule.

    1. Dispersive Model

    • Proposes that each strand of both daughter molecules contains a mixture of old and newly synthesised DNA.

    • The parental DNA molecule breaks up into short segments, which act as templates for synthesizing DNA.

    • The segments are then joined together, resulting in both old and new DNA interspersed along each strand in both daughter DNA molecules.

    1. Semi-Conservative Model (Watson & Crick’s DNA model)

    • Proposes that the double helix parental molecule replicates and each of the daughter molecules consists of one parental strand and one newly synthesised strand.

    • The two strands of the parental molecule separate, and each strand acts as a template for synthesizing a newly synthesised strand.

    • Each daughter molecule contains one strand conserved from the parental molecule and one newly synthesised strand.

Semi-Conservative Replication

  • During cell division, DNA replication is essential for ensuring each daughter cell has a complete set of genes.

  • Occurs in the synthesis phase of interphase during both mitotic and meiotic cell cycles.

Step 1: Unwinding

  • DNA replication begins at a specific sequence of nucleotides known as the origin of replication.

  • DNA helicase unwinds and unzips a portion of the double helix, breaking hydrogen bonds between nitrogenous bases as it moves along the DNA.

    • Uses energy from ATP to facilitate the unwinding.

  • As parental strands separate, a replication bubble forms, creating two replication forks.

  • In prokaryotic chromosomes, there is a single origin of replication, whereas eukaryotic chromosomes have multiple origins, leading to multiple replication bubbles that eventually fuse.

Step 2: Priming

  • Short segments of RNA, typically 5 to 10 nucleotides in length, known as RNA primers, are synthesized to initiate replication.

  • Primase, a specialized RNA polymerase, binds to the single-stranded DNA template and synthesizes RNA primers in the 5' to 3' direction.

    • These primers provide the necessary 3’ OH end for DNA polymerase to initiate elongation.

Step 3: Elongation

  • Before replication starts, free deoxyribonucleotides are synthesised in the cytoplasm and transported to the nucleoplasm via nuclear pores.

  • DNA polymerase adds deoxyribonucleotides (more specifically, deoxyribonucleoside triphosphates, dNTPs) to the 3’ OH end of the RNA primer.

  • The formation of a phosphodiester bond occurs between the 3' OH end of the primer and the 5' phosphate group of the dNTP added.

  • The DNA strand is synthesized in the 5' to 3' direction based on complementary base pairing with the parental strand.

    • The base pairs are A=T and G≡C.

  • Due to the antiparallel nature of DNA strands:

    • Leading Strand is synthesized continuously towards the replication fork. Only one primer is needed.

    • Lagging Strand is synthesized discontinuously, forming Okazaki fragments, requiring multiple primers and resulting in segments that are joined together later.

Step 4: Termination

  • The replication process generates two daughter DNA molecules, each identical to the original parental molecule, each containing one strand conserved from the parental molecule and one newly synthesised strand.

Transcription

  • Transcription refers to the synthesis of an RNA molecule using one DNA strand as a template and occurs in three main stages:

    1. Initiation

    2. Elongation

    3. Termination

Overview of Transcription

  • RNA synthesis goes from the 5’ end to the 3’ end. The synthesised RNA strand is similar to the non-template DNA strand, being complementary to the template DNA strand.

Types of RNA Synthesised

  • The main types of RNA synthesized during transcription are:

    • Messenger RNA (mRNA)

    • Transfer RNA (tRNA)

    • Ribosomal RNA (rRNA)

Initiation of Transcription

  • In prokaryotes, the sigma factor of RNA polymerase recognizes and binds to the double-stranded DNA at the -35 and -10 consensus sequences of the promoter, where the -10 sequence is the Pribnow box (5' – TATAAT – 3').

  • RNA polymerase unwinds and separates the DNA strands, exposing the template strand for complementary base pairing with ribonucleotides.

  • In eukaryotes, the TATA binding protein (TBP) binds to the TATA box of the promoter (located 25 base pairs upstream of the transcription start site). Transcription factors and RNA polymerase form the transcription initiation complex.

Elongation of Transcription

  • RNA polymerase synthesizes RNA nucleotides in the 5' to 3' direction, forming phosphodiester bonds.

  • The energy for RNA synthesis is derived from the hydrolysis of nucleoside triphosphates (NTPs). A short RNA-DNA hybrid is also formed during this process.

Termination of Transcription

  • In prokaryotes, transcription terminates after a terminator sequence is reached, releasing the RNA transcript without further modification needed.

  • In eukaryotes, the termination involves a polyadenylation signal sequence (AAUAAA), resulting in pre-mRNA cleaving and subsequent post-transcriptional modifications before transport to cytoplasm via nuclear pores.

Translation

  • Translation is the synthesis of protein using mRNA as a template and also occurs in three main stages:

    1. Initiation

    2. Elongation

    3. Termination

Amino Acid Activation

  • Before translation, amino acids need activation by corresponding tRNAs. This process involves covalently attaching the amino acid to the 3’ end of the specific tRNA by an enzyme called aminoacyl-tRNA synthetase, which is driven by ATP hydrolysis.

Paring of Codon and Anticodon

  • tRNA anticodon must match with mRNA codon, where variations in the third base are accommodated due to wobble effect, allowing fewer tRNAs than codons.

Initiation of Translation

  • The small ribosomal subunit binds to the 5’ end of mRNA and the initiator tRNA, scanning for the start codon (AUG) to establish the reading frame. The large ribosomal subunit then associates, completing the translation initiation complex.

Elongation of Translation

  • Amino acids are sequentially added to the peptide chain through peptide bond formation catalyzed by the large ribosomal subunit. The ribosome translocates, moving along the mRNA.

Termination of Translation

  • Termination occurs when a stop codon (UAG, UAA, UGA) reaches the A site of the ribosome, leading to hydrolysis of the polypeptide from the tRNA and the disassembly of the translation complex. Energy from GTP hydrolysis is needed during this process.

Conclusion

  • This lesson on molecular genetics covers the processes of DNA replication, transcription, and translation, outlining the models, steps, and enzymes involved in each stage to ensure the accurate expression of genetic information.

Prepared by: Mel Tan