PH1123 UNIT 2, lesson 1 PART 2 nucliec acids to protiens

• The Central Dogma of Molecular Biology describes the flow of genetic information from DNA to protein, via RNA It consists of three main processes:
  • Replication (DNA → DNA)
  • Transcription (DNA → RNA)
  • Translation (RNA → Protein)
• Replication prepares the DNA by copying it before cell division, ensuring each new cell has a complete set of genetic instructions
• It occurs during the S-phase of the cell cycle
• DNA polymerase is the key enzyme involved
• Transcription, the first step of the Central Dogma, involves RNA polymerase transcribing DNA into mRNA after replication
• Translation, the final step, involves mRNA being decoded into a protein by ribosomes
• The genetic code from DNA is expressed as functional proteins

Functional Proteins

• After translation, proteins are synthesized and can perform diverse functions in the cell:
  • Enzymes: Catalyze biochemical reactions, speeding up chemical reactions in the cell; examples include DNA polymerase (replicates DNA) and RNA polymerase (transcribes DNA to RNA)
  • Structural Proteins: Provide mechanical support and structure
    • Examples include collagen (connective tissue) and keratin (hair, nails, skin)
  • Transport Proteins: Facilitate molecule transport, helping move substances within the body or across cell membranes
    • Examples include hemoglobin (carries oxygen in blood) and ion channels (move ions across membranes)
  • Regulatory Proteins: Control gene expression
    • Example is transcription factors, regulate DNA transcription and protein synthesis
  • Signaling Proteins: Mediate communication between cells
    • Example- Hormones like insulin, transmit signals for cellular responses
  • Immune Proteins: Defend against pathogens
    • Example- Antibodies, protect the body from infections
  • Motor Proteins: Enable movement
    • Examples include myosin and actin (involved in muscle contraction)

DNA Replication

• In cell division, the DNA molecule is replicated so that each daughter cell will carry its own DNA molecule
• DNA replication proceeds in three enzymatically mediated steps:
  • initiation, elongation, and termination
• DNA replication is initiated in DNA regions known as “origins” and starts by unwinding the DNA double strand
• This process uses helicases, which break the hydrogen bonds that 'bind' the DNA strands together
• Each parent strand acts as a template for the synthesis of a new DNA strand; the new DNA strands are complementary to the parent strands
• DNA synthesis is catalyzed by DNA polymerases and is synthesized from 5' to 3'
• Termination is achieved by blocking the replication fork

DNA Replication Fork

• The Y-shaped structure that forms when DNA is being replicated is known as the replication fork
• It is created when helicase unwinds the double-stranded DNA, separating it into two single strands to serve as templates for new DNA synthesis
• Key Features of the Replication Fork: Leading and Lagging Strands
  • The leading strand is synthesized continuously in the 5' to 3' direction
• The lagging strand is synthesized discontinuously, resulting in Okazaki fragments because polymerase can only add nucleotides in the 5' to 3' direction
• Enzymes Involved:
  • Helicase: Unwinds the DNA strands
  • Single-strand binding proteins (SSBs): Prevent separated strands from reannealing
  • Topoisomerase: Relieves supercoiling tension ahead of the fork Primase: Synthesizes RNA primers to initiate replication
  • DNA Polymerase: Adds nucleotides to synthesize new strands
  • DNA Ligase: Joins Okazaki fragments on the lagging strand
• DNA replication is bidirectional, so two replication forks form at the origin, efficiently copying the entire DNA molecule

DNA Replication Steps

• Initiation
  • The process begins at specific sites called origins of replication
  • Helicase unwinds the double-stranded DNA, breaking hydrogen bonds creating a replication fork
  • Single-strand binding proteins (SSBs) attach to the separated strands
  • Topoisomerase prevents supercoiling ahead of the replication fork
  • Primase synthesizes a short RNA primer to provide a starting point for DNA polymerase
• Elongation
  • DNA polymerase adds complementary nucleotides to the growing DNA strand in the 5' to 3' direction
  • The leading strand is synthesized continuously in the direction of the replication fork
  • The lagging strand is synthesized in short segments called Okazaki fragments, which are later joined by DNA ligase
• Termination
  • Replication continues until the entire DNA molecule is copied
  • In prokaryotes, replication ends at specific termination sites (ter sites)
  • In eukaryotes, replication stops when replication forks meet or when telomeres are reached
  • The enzyme telomerase in eukaryotes helps maintain telomere length to prevent chromosome degradation
  • RNA primers are replaced with DNA, with DNA ligase sealing any remaining gaps

dNTPs in DNA Replication

• Precursors:
  • The precursors for the synthesis of new DNA strands are nucleoside triphosphates (dNTPs), like dATP, dTTP, dGTP, and dCTP
  • Triphosphate anhydrides that attacks hydroxyl groups
• DNA Chain Extension:
  • DNA chain extension involves an esterification reaction of the 3'-hydroxyls, with triphosphate anhydrides acting as a good leaving group as a diphosphate
  • The correct triphosphate is selected based on the hydrogen bonding properties of the base pairs
• The steps of dNTPs in DNA Replication are:
  1. dNTP is available in the cell
  2. DNA polymerase selects the correct dNTP based on base pairing (A-T, C-G)
  3. OH of the growing DNA strand attacks the first phosphate of the dNTP, resulting in phosphodiester bond
  4. Two phosphates releases for energy
  5. DNA strand extends, and the process repeats

Nucleosides

• Nucleotides have 3 phosphates before they're incorporated into the DNA chain
• Once incorporated, two of the phosphates are "split off" during the reaction
• The one phosphate is incorporated into the DNA chain and connects to the sugar of the next nucleotide.
• dNTPs act as building blocks for DNA synthesis
• During replication, DNA polymerase removes two phosphates (pyrophosphate) from the triphosphate, converting it into nucleoside monophosphate (NMP) and attaching it to the growing DNA strand.

RNA

• Structural Differences
  • RNA differs structurally from DNA in three important ways:
    • The sugar in RNA is ribose, while in DNA it's deoxyribose
    • Thymine is replaced by uracil in RNA
    • RNA is typically single-stranded
• RNA stores genetic information and participates in the processes by which this information is used
• Three major forms of RNA are found in prokaryotic cells:
  • mRNA, tRNA, and rRNA

Transcription

• Transcription makes RNA through, after DNA is replicated
• RNA polymerase synthesizes a complementary RNA strand from the DNA template strand, using ribonucleotides (ATP, CTP, GTP, UTP) to build mRNA
• RNA is single-stranded and contains uracil (U); the mRNA sequence is complementary to the DNA template strand but identical (except U instead of T) to the coding strand
• This process occurs in the nucleus (in eukaryotes)

Genetic Code

• The genetic code is read three nucleotides at a time
• it is the sequence of bases along one DNA strand, the “coding” strand (a.k.a. the Messenger Ribonucleic acid - mRNA)
• It carries the information to build a template strand, the segment that is necessary for the synthesis of one protein
• Each amino acid in a protein is specified by any sequence of three nuclides called codon

Messenger RNA Synthesis

• mRNA doesn't have to use Okazaki fragments unlike DNA
• No RNA Primers are used
• Although the amino acid sequence of a protein is defined by the sequence of codons in DNA, it is RNA that participates in the interpretation of this sequence
• The synthesis of mRNA from the DNA template is called transcription
• First, DNA is unwound and the template strand is the one used for the mRNA synthesis
• U is used instead of T
• Transcription occurs in the nucleus

Transfer RNA

• After synthesis, mRNA moves from the nucleus to the cytoplasm and ribosome
• Ribosomes (site of protein synthesis, combination of RNA + protein) are made up of 2 subunits termed S50 and S30
• Transfer RNA (tRNA) + Amino acid will become charged tRNA after Acylation
• A tRNA is specific for a particular amino acid, one arm of tRNAs, at 3’ end, will always have to which amino acids are ligated

Translation Key Players

• mRNA (Messenger RNA) – Carries the genetic code from DNA
• tRNA (Transfer RNA) – Brings amino acids to the ribosome
• Ribosome – The site of protein synthesis, made of rRNA + proteins

Translation Steps

1. Initiation- Ribosome + 1st tRNA that carries Met binds on the mRNA at start codon
2. Elongation
   • New tRNA enters A site
   • Ribosome forms peptide bond between A site and P site
   • Shifting occurs
     • Empty tRNA exits from E site
     • Growing polypeptide moves to the P side
     • New tRNA enter A site and the process begins again
3. Termination- When the ribosome reaches a stop codon, a protein release factor happens

tRNA Acylation

• Acylation, the attachment of an amino acid to tRNA at its 3'-OH group forming an ester bond
• It’s SN2, happens in 2 steps, and needs 1 ATP

1. The amino acid reacts with ATP, forming an Aminoacyl-AMP intermediate
2. The OH attacks the carboxyl, forms Aminoacyl-AMP(nucleophilic substitution)

Protein Synthesis

• The mRNA sequence is read from the 5' to the 3' in translation
• In prokaryotes, the first amino acid encoded is N-formylmethionine, which has the same codon as methionine (AUG)
• Note that the peptide is synthesized from the N (amino) terminal to the C (carboxyl) terminal

Protein Terminals

• N-Terminus (Amino Terminus) is the Start of the polypeptide, a free Amino group
• C-Terminus (Carboxyl Terminus) is the End of the polypeptide, a free Carboxyl group

Translation Steps in Terminal Terms

• Proteins are synthesized from N-terminus to C-terminus
• The ribosome reads mRNA from 5' to 3', adding new amino acids to the C-terminal end
• Peptide bonds form between the C-terminal (-COOH) of one amino acid and the N-terminal (-NH2) of the next.