Pre-mRNA Processing and DNA Replication

• DNA replicates semiconservatively
• The parental DNA molecule separates into two strands, and each acts as a template
• This allows synthesis of a new, complementary strand
• The replicated DNA consists of one old and one newly made strand

DNA Strand Separation

• DNA replication starts at replication origins
• DNA helicase binds to these origins and breaks hydrogen bonds
• This unwinds the DNA, forming a replication "bubble" with two replication forks
• Replication forks form at the junction of single and double-stranded regions
• Single-stranded binding proteins prevent reformation of the double helix

Synthesis of RNA Primer

• Both parental strands act as templates for synthesizing new DNA strands
• Primase synthesizes an RNA primer where replication begins
• Synthesis occurs in the 5' to 3' direction
• RNA nucleotides are added complementary to the DNA
• An RNA primer, comprised of 5 to 10 RNA nucleotides, forms on each strand

DNA Synthesis Direction

• DNA polymerase III catalyzes new DNA synthesis by adding DNA nucleotides to the 3' end of an existing chain
• This enzyme also catalyzes the phosphodiester linkage between DNA nucleotides
• New DNA strands are therefore synthesized in the 5' to 3' direction
• The added DNA nucleotides are complementary to the template strand (A-T, G-C)

Leading and Lagging Strands

• Two types of new strands exist because DNA strands are antiparallel: leading and lagging
• DNA nucleotides are added continuously in the leading strand, growing towards the replication fork
• DNA nucleotides are added discontinuously in the lagging strand in segments called Okazaki fragments, growing away from the fork
• Each Okazaki fragment is initiated by a separate RNA primer

Replacement of RNA Primer

• DNA polymerase I removes RNA nucleotides of the primer and replaces them with DNA nucleotides

Ligation of DNA Fragments

• DNA ligase joins Okazaki fragments on the lagging strand, forming phosphodiester linkages
• This results in two daughter DNA molecules with one old and one new strand each

Protein Synthesis

• Gene expression, directs protein synthesis (or RNAs)
• It encompasses two stages: transcription and translation

Transcription

• Produces RNA using DNA's information
• The DNA double helix separates into a template/antisense strand (which is transcribed) and a coding/sense strand (not transcribed)
• RNA is synthesized in the 5'-3' direction without needing a primer
• Bacteria use one type but eukaryotes have three types of RNA polymerase
• RNA polymerase I synthesizes rRNA, RNA polymerase II synthesizes mRNA, and RNA polymerase III synthesizes tRNA
• Transcription consists of initiation, elongation, and termination

Initiation

• It starts at the promoter, the binding site of RNA polymerase on the template strand
• Transcription factors bind to the promoter, which enables RNA polymerase binding
• RNA polymerase unwinds and separates the DNA helix

Elongation

• RNA polymerase moves along the template strand, unwinding DNA and exposing 10-20 DNA nucleotides for pairing with RNA nucleotides
• Ribonucleotides are added to the 3' end of the RNA strand, complementary to the template
• A in DNA pairs with U in RNA, C in DNA pairs with G in RNA, and T in DNA pairs with A in RNA
• As RNA elongates, the newly formed RNA strand separates from the DNA template and the DNA template strand rewinds, reforming the double helix

Termination

• RNA polymerase reaches a 'stop' signal
• The newly formed RNA and DNA separate
• DNA rewinds ending transcription

Post-Transcriptional Modification

• Translation begins in prokaryotes while transcription is still in progress
• This is possible due to the lack of a nuclear membrane in prokaryotes, allowing bacterial DNA and ribosomes to be in the same cytoplasm
• Eukaryotes require modification of pre-mRNA before it exits the nucleus, preventing degradation in the cytoplasm
• This modification occurs only in eukaryotes

mRNA modification

• A modified form of guanine nucleotide is added as a 5' cap.
• Around 50-250 adenine nucleotides are added at the 3' end, forming a poly-A tail.

RNA Splicing

• Eukaryotic genes consist of exons (coding segments) and introns (non-coding segments)
• Initial transcription produces a pre-mRNA transcript, containing both
• Spliceosomes cut out introns and join exons to form shorter, mature mRNA transcript.

The Genetic Code

• There are 64 codons which is the language for protein synthesis
• A codon consists of a sequence of 3 consecutive mRNA bases
• The code is read continuously

Degeneracy

• The genetic code is degenerate; more than one codon can specify a single amino acid
• No codon specifies more than one kind of amino acid
• Certain codons act as 'start' (AUG, coding for methionine) or 'stop' (UAA, UAG, UGA) signals
• It is nearly universal, can be used across both the simplest bacteria and complex animals.

Translation

• The synthesis of a polypeptide using the genetic information in mRNA
• It involves a change of "language" from nucleotides to amino acids with tRNA serving as the translator
• Ribosomes provide a site for polypeptide assembly

Ribosomes

• Composed of large and small subunits, made of rRNA and protein
• Have three sites for protein synthesis: P, A, and E
• Also has an mRNA binding site

tRNA Function

• Transfers amino acids from the cytoplasm to ribosomes
• At the opposite end, a specific amino acid is attached by aminoacyl-tRNA synthetase which create aminoacyl-tRNA complexes
• There is no tRNA for 'nonsense codons'

Translation Stages

• There are 3 key stages in translation: initiation, elongation, and termination

Initiation (Translation)

• Small ribosomal subunit binds to mRNA at the AUG start codon.
• Initiator tRNA (methionine-tRNA) with anticodon UAC binds to AUG.
• The large ribosomal subunit then binds to the small subunit
• This completes the initiation complex, with initiator tRNA at the P site, and A & E sites empty.

Elongation (Translation)

• A 2nd tRNA, carrying a complementary anticodon and attached amino acid, binds at the A site
• Peptidyl transferase then breaks the bond between the amino acid and the tRNA at the P site
• It then attaches this amino acid to the amino acid at the A site, creating a peptide bond
• The 1st tRNA is now empty, and the 2nd tRNA has 2 amino acids
• The ribosome then moves one codon in the 5' to 3' direction
• The 1st tRNA shifts to the E site, 2nd tRNA shifts to the P site, and the A site is left empty
• A 3rd tRNA with an amino acid now binds the A site
• Peptidyl transferase then breaks the bond holding the 2nd amino acid to the 2nd tRNA, attaching it to the 3rd amino acid
• The 2nd tRNA is now empty, and the 3rd tRNA has 3 amino acids
• The 1st tRNA departs the ribosome, which shifts another codon, repeating the process

Termination (Translation)

• Release factor binds to the stop codon at the A site
• Release factors promote hydrolysis, releasing the polypeptide releasing the polypeptide
• Ribosomal subunits and other components then dissociate

Gene Expression Differences

• In prokaryotes, RNA is translated as soon as it is transcribed but this is not true for eukaryotes
• Prokaryotic genes do not contain introns, they do not undergo RNA splicing, and the RNA is not modified
• In eukaryotes, RNA must be transported, the genes contain introns, RNA splicing happens, and the RNA is modified

Gene Expression Control

• Genes are regulated, so only the genes directly needed are transcribed
• Some encode constant proteins termed constitutive genes
• Some genes are directed based on need

Operons

• Gene expression regulation in prokaryotes
• It's a unit of genetic function found in bacteria and phages
• It consists of a promoter, an operator, and structural genes
• The promoter the sequence where RNA polymerase binds
• The operator is a switch that controls transcription
• Structural genes are a cluster of genes coordinately regulated

Operon Details

• The operator is regulated by a repressor produced by a regulatory gene.
• An active repressor will bind to the operator, blocking the promoter and preventing RNA polymerase from binding, thus preventing transcription

Inducible Operons (lac Operon)

• Usually off, switched on in the presence of an inducer
• When both glucose and lactose are available, E. coli up all the glucose first
• Once glucose is used up, it will use the lactose to form glucose and galactose
• E. coli needs genes to hydrolyze the enzymes

Lac-Operon

• The Lac operon of E. coli controls production of enzymes that hydrolyze lactose.
• Its components include a lac operator (O), lac Promoter (Plac) and structural genes (lacZ, lacy, lacA)
• Beta-galactosidase hydrolyzes lactose, permease transports lactose, and transacetylase detoxifies molecules that enter the cell

Absence of Lactose

• lac repressor (active) binds to the lac operator blocking part of the lac promoter
• Due to this, RNA cannot bind
• Thus, transcription is repressed

Presence of Lactose

• Some lactose converts to allolactose which binds the repressor
• The shape changes and the repressor cannot bind
• The promoter is unblocked
• RNA binds and transcription begins

Explore pre-mRNA processing steps like 5' capping and intron removal. Learn about mRNA codons, the genetic code, and modifications such as the poly-A tail. Understand the roles of key enzymes in DNA replication.