From DNA to Protein: A Deep Dive into Protein Synthesis

Posted on Aug 23, 2024 in Biology

The Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is an artificial method of replicating DNA under laboratory conditions. The process involves the following steps:

  1. Denaturation: The DNA sample is heated (~90ºC) to separate the two strands.
  2. Annealing: The sample is cooled (~55ºC) to allow primers to anneal. Primers designate the specific sequence to be copied.
  3. Elongation: The sample is heated to the optimal temperature for a heat-tolerant polymerase (Taq) to function (~75ºC). Taq polymerase extends the nucleotide chain from the primers, thereby selecting the sequence to be copied.

DNA Structure

DNA is composed of two strands. Each strand has a phosphate group attached to the 5′ carbon of the deoxyribose sugar at one end (5′ terminal) and a hydroxyl group attached to the 3′ carbon at the other end (3′ terminal). The strands run in opposite directions, making them antiparallel. One strand runs in a 5′-3′ direction, while the other runs in a 3′-5′ direction.

Adjacent nucleotides are linked by a phosphodiester bond between the phosphate group of one nucleotide and the 3′ carbon of the deoxyribose of the next nucleotide. The bases of each strand pair with each other via hydrogen bonds.

Purines (Adenine and Guanine) have two rings in their molecular structure, while pyrimidines (Thymine and Cytosine) have one. In base pairing, a purine always pairs with a pyrimidine. Adenine pairs with thymine via two hydrogen bonds, while guanine pairs with cytosine via three hydrogen bonds.

DNA Replication

Key Enzymes

  • Helicase: Unwinds the DNA double helix by breaking the hydrogen bonds between complementary base pairs, creating two template strands for replication.
  • DNA Polymerase: Synthesizes new DNA strands from the parental template strands by adding free deoxynucleoside triphosphates (dNTPs) to the 3′ end of the growing strand. The energy for this process comes from the hydrolysis of two phosphate groups from the dNTPs.

DNA Replication in Prokaryotes (HL)

DNA replication is semi-conservative, meaning each new DNA molecule consists of one original and one newly synthesized strand. The process in prokaryotes involves the following steps:

  1. Uncoiling: Helicase unwinds the DNA double helix.
  2. Primer Synthesis: RNA primase adds a short RNA primer to the template strands, providing a starting point for DNA polymerase III.
  3. Elongation: DNA polymerase III adds nucleotides to the 3′ end of the primer, synthesizing the new strand in a 5’→3′ direction. The leading strand is synthesized continuously, while the lagging strand is synthesized in fragments called Okazaki fragments.
  4. Primer Removal and Gap Filling: DNA polymerase I removes the RNA primers and replaces them with DNA nucleotides.
  5. Joining of Fragments: DNA ligase joins the Okazaki fragments to form a continuous strand.

Transcription

Transcription is the process of synthesizing RNA from a DNA template. The steps involved are:

  1. Initiation: RNA polymerase binds to the promoter region of the DNA.
  2. Elongation: RNA polymerase unwinds the DNA helix and adds RNA nucleotides complementary to the template strand (antisense strand), synthesizing the RNA molecule in a 5’→3′ direction.
  3. Termination: RNA polymerase reaches the terminator sequence, signaling the end of transcription. The RNA molecule is released, and the DNA helix rewinds.

Key Points

  • The mRNA molecule is a single-stranded copy of the DNA’s coding sequence (sense strand), with uracil (U) replacing thymine (T).
  • The antisense strand serves as the template for mRNA synthesis.

Translation

Translation is the process of synthesizing proteins from mRNA. It occurs in the cytoplasm and involves the following steps:

  1. Initiation: A tRNA carrying methionine (anticodon UAC) binds to the start codon (AUG) on the mRNA at the ribosome’s P site.
  2. Elongation: The next tRNA with the matching anticodon binds to the A site. A peptide bond forms between the amino acids on the two tRNAs. The ribosome translocates, moving the first tRNA to the E site (exit) and the second tRNA to the P site, allowing the next tRNA to bind.
  3. Termination: The ribosome reaches a stop codon (UAA, UAG, or UGA) on the mRNA. No tRNA has a matching anticodon, so the polypeptide chain is released.

tRNA and Amino Acid Activation

  • Each tRNA molecule carries a specific amino acid and has a corresponding anticodon that recognizes a specific codon on the mRNA.
  • tRNA-activating enzymes attach the correct amino acid to their corresponding tRNA molecules using ATP.

Polysomes

Multiple ribosomes can translate the same mRNA molecule simultaneously, forming a complex called a polysome. This allows for efficient protein synthesis.

Differences Between Eukaryotes and Prokaryotes

Eukaryotes

  • Transcription occurs in the nucleus, while translation occurs in the cytoplasm.
  • mRNA undergoes modifications (5′ capping and 3′ polyadenylation) before leaving the nucleus.

Prokaryotes

  • Lack a nucleus, so transcription and translation can occur simultaneously in the cytoplasm.
  • Ribosomes can begin translating mRNA while it is still being transcribed.

Protein Destination

is determined by the presence or absence of an initial signal sequence on a nascent polypeptide chain.

1.The presence of this signal sequence results in the recruitment of a signal recognition particle (SRP), which halts translation.

2.The SRP-ribosome complex then docks at a receptor located on the ER membrane (forming rough ER)

Translation is re-initiated and the polypeptide chain continues to grow via a transport channel into the lumen of the ER.The synthesised protein will then be transported via a vesicle to the Golgi complex (for secretion) or the lysosome. Proteins targeted for membrane fixation (e.g. integral proteins) get embedded into the ER membrane. The signal sequence is cleaved and the SRP recycled once the polypeptide is completely synthesised within the ER.