DNA Replication, Transcription, and Translation

DNA Polymerase Principles

• DNA polymerase synthesizes in the 3' to 5' direction.
• Requires pre-existing nucleic acid at the 5' site for nucleotide attachment.
• Encounters issues connecting new nucleotides to existing nucleic acid.

DNA Replication In Vivo

• Occurs during the S phase of the cell cycle.
• Initiates at specific regions called origins of replication (ORI).
• Bacteria: single, circular chromosome with one ORI.
• Eukaryotes: multiple origins of replication activated simultaneously on each chromosome.

Origin of Replication

• DNA is initially double-stranded.
• Needs to be unwound into single-stranded DNA for polymerase access.
  • One strand runs 5' to 3', the other runs 3' to 5'.

DNA Helicase

• Enzyme responsible for denaturing (unzipping) the DNA double helix at the origin of replication.
• Two helicases bind at each origin and move in opposite directions.
• Forms a replication bubble of single-stranded DNA, allowing DNA polymerase interaction.

Replication Fork

• The structure formed as DNA is unzipped by helicase.

DNA Polymerase III

• Specific DNA polymerase involved in replication.
• Requires a pre-existing nucleic acid strand to add nucleotides.

Priming

• RNA primase synthesizes a short, temporary RNA primer.
• Provides a 3' end for DNA polymerase to attach new nucleotides.
• DNA polymerase III attaches to the primer and extends it with DNA nucleotides in the 3' to 5' direction along the template strand.

Leading Strand

• DNA polymerase follows the DNA helicase continuously.
• Requires only one initial RNA primer for synthesis.

Lagging Strand

• DNA polymerase moves in the opposite direction of the replication fork.
• Requires repeated initiation of RNA primers as the helicase unzips more DNA.
• Results in discontinuous synthesis.

Origin of Replication Details

• Replication proceeds bidirectionally from the origin.
• At each replication fork, one strand is the leading strand and the other is the lagging strand.
• The leading strand is synthesized continuously following the helicase.
• The lagging strand requires repeated initiation and synthesis in short fragments because it's going in the opposite direction from the helicase.
• The DNA polymerase will add nucleotides until it reaches a previously synthesized RNA primer.

Replication Results

• The end result is a new strand with RNA primers interspersed with newly synthesized DNA.

DNA Polymerase I

• Enzyme that removes RNA primers and replaces them with DNA nucleotides.
• Possesses exonuclease activity to cut out RNA and polymerase activity to add DNA.
• It moves along the template in the 3' to 5' direction, replacing RNA with DNA.
• Cannot form the final phosphodiester bond to connect the DNA fragments.

DNA Ligase

• Enzyme that seals the gaps (nicks) between the DNA fragments after RNA primer removal and replacement.
• Forms the final phosphodiester bond to create a continuous strand of DNA.

Semi-Conservative Replication

• Each new DNA molecule consists of one original (template) strand and one newly synthesized strand.

Practical Application of Enzymes

• Restriction enzymes cut DNA at specific sequences.
• DNA ligase can join DNA fragments from different sources.
• Example: Inserting a human insulin gene into a bacterial plasmid, creating a recombinant DNA molecule.
• The genetically modified bacteria can then produce human insulin.

Historical Context

• Previously, insulin was extracted from livestock pancreases.
• Livestock insulin differs slightly from human insulin, potentially causing allergic reactions in patients.
• Recombinant human insulin produced by bacteria is identical to human insulin, reducing the risk of allergic reactions.

Transcription

• Making an RNA complement of DNA.

Gene Definition

• Area of DNA that is transcribed.

Chromosome Structure

• Genes are located on chromosomes.
• Eukaryotic chromosomes contain large non-coding regions between genes.
• The function of these regions is still under investigation.

Gene Types

• Protein-coding genes (mRNA).
• tRNA genes (tRNA).
• rRNA genes (rRNA).

mRNA

• Messenger RNA; carries instructions for protein synthesis to the ribosome.

tRNA

• Transfer RNA; transports amino acids to the ribosome for protein synthesis.

rRNA

• Ribosomal RNA; a component of the ribosome structure itself.
• Most abundant type of RNA in the cell.

Differential Gene Expression

• Not all genes are transcribed at the same time or in every cell type.
• Example: Collagen genes are expressed in skin cells, while insulin genes are expressed in pancreatic cells.
• tRNA and rRNA genes are generally transcribed in all cell types.

Eukaryotic Protein-Coding Genes and Transcription

• Promoter: DNA sequence that initiates transcription; has directionality.
• RNA polymerase: Enzyme that synthesizes RNA in the 3’ to 5’ direction along the template DNA.
• Terminator: DNA sequence that signals the end of transcription.
• UTRs (Untranslated Regions): Regions at the 5’ and 3’ ends of mRNA that are not translated into protein.
• Exons: Coding regions of a gene that contain amino acid sequence information.
• Introns: Non-coding regions within a gene that are removed during RNA processing.

Transcription Process

• RNA polymerase binds to the promoter and synthesizes a pre-mRNA molecule.
• pre-mRNA contains UTRs, exons, and introns.

Strand Terminology:

• Template strand (non-coding strand).
• Coding strand.

RNA Processing (occurs in the nucleus):

• Splicing: Removal of introns from the pre-mRNA by the spliceosome.
• Spliceosome: Complex of proteins and RNA that recognizes intron-exon boundaries and removes introns.
• 5’ cap: Addition of a modified guanine nucleotide to the 5’ end of the mRNA.
• Poly-A tail: Addition of a string of adenine nucleotides to the 3’ end of the mRNA.

Mature mRNA:

• Contains the 5’ cap, 5’ UTR, exons, 3’ UTR, and poly-A tail.
• The 5’ cap and poly-A tail protect the mRNA and signal for its export from the nucleus.

Alternative Splicing:

• Different combinations of exons and introns can be removed, resulting in different mRNA molecules from the same gene.
• Allows for the production of multiple protein isoforms from a single gene.

Translation

• Also known as protein synthesis.

Ribosome Structure:

• Small subunit.
• Large subunit: Contains the A (aminoacyl), P (peptidyl), and E (exit) sites.

Initiation:

• The small subunit binds to the 5’ end of the mRNA and scans for the start codon (AUG).

• tRNA with methionine (Met) binds to the start codon in the P site.

• The large subunit joins the complex, forming the functional ribosome.

• Codon: A sequence of three mRNA nucleotides that specifies a particular amino acid (or a stop signal).

• The genetic code is read in codons during translation.

tRNA Details:

• Each tRNA molecule is specific to one amino acid.
• Aminoacyl-tRNA synthetases attach the correct amino acid to its corresponding tRNA.
• The anticodon on the tRNA base-pairs with the codon on the mRNA.