Functions of Genetic Material

Functions of Genetic Material

• DNA carries genetic information and is passed down from parents to offspring in all living organisms
• DNA replication is highly accurate, minimizing sequence changes

DNA Function

• Directs and regulates protein construction for cellular growth and reproduction

Gene Expression

• The process of synthesizing a specific protein from the amino acid sequence encoded in a gene
• The central dogma describes the flow of genetic information from DNA to RNA to protein

DNA Replication Models

• Conservative model: parental DNA strands remain together, and new daughter strands form a separate molecule
• Semiconservative model: parental strands separate and each directs the synthesis of a daughter strand creating hybrid molecules
• Dispersive model: resulting DNA strands contain both parental and daughter DNA regions

Experimental Verification

• Meselson and Stahl used E. coli grown in heavy nitrogen (15N) and then in normal nitrogen (14N)
• DNA with 15N was denser and sedimented lower during ultracentrifugation
• After one replication round, DNA sedimented halfway between 15N and 14N levels, disproving the conservative model
• After a second round, the dispersive model was disproved, supporting semiconservative replication

DNA Polymerase III

• Adds deoxyribonucleotides to the 3'-OH group of a growing DNA chain, complementary to the template strand
• Requires energy from the bonds of the triphosphate nucleotide, similar to ATP

Energy Source

• The bonds of the three phosphate groups attached to each nucleotide (a triphosphate nucleotide)
• Breaking the bond between phosphates releases energy for phosphodiester bond formation

Key Enzymes

• Topoisomerase II relaxes supercoiled chromosomes
• Helicase separates DNA strands
• Single-stranded binding proteins keep strands separated
• RNA primase synthesizes an RNA primer
• DNA polymerase III elongates the primer
• DNA polymerase I removes RNA primers
• DNA ligase joins Okazaki fragments

Replication Fork and Direction

• Replication forks form at the origin of replication, proceeding bidirectionally
• DNA replication occurs in both directions

Primer Synthesis

• RNA primer synthesized by RNA primase is complementary to the parental strand
• Primer is elongated by DNA polymerase III, adding nucleotides to the 3'-OH end

Leading vs Lagging Strand

• Leading strand: synthesized continuously
• Lagging strand: synthesized in short Okazaki fragments

Okazaki Fragments

• Short DNA stretches synthesized on the lagging strand
• RNA primers are removed by DNA polymerase I
• Okazaki fragments are joined by DNA ligase

Origin of Replication

• Initiation of replication occurs at a specific nucleotide sequence
• E. coli has a single origin of replication (oriC) on its chromosome

Sequences

• 245 base pairs long
• Rich in adenine-thymine (AT) sequences

Bacterial DNA Replication Steps

• Supercoiled chromosome relaxed by topoisomerase II (DNA gyrase)
• Helicase separates DNA strands by breaking hydrogen bonds between base pairs
• Y-shaped replication forks are formed

Replication Bubble

• Structure formed with two replication forks at the origin
• Prevents rewinding of single-stranded DNA into a double helix via single-stranded binding proteins

DNA Polymerase III Directionality

• Nucleotides are added only in the 5' to 3' direction, requiring a free 3'-OH group
• DNA and RNA polymerases do not need a free 3'-OH group to synthesize an RNA molecule

Elongation Rate

• Nucleotides are added at a rate of about 1000 nucleotides per second

Strand Orientation

• The DNA double helix is antiparallel (one strand is 5' to 3', the other is 3' to 5')
• The continuously synthesized strand toward the replication fork is the leading strand

Synthesis of Strand

• The strand growing away from the replication fork requires the polymerase to move back to add new primers.
• This produces Okazaki fragments
• The lagging strand synthesis is discontinuous

Sliding Clamp

• Ring-shaped protein that binds to the DNA and holds polymerase in place
• Prevents overwinding

Primers Replacement

• RNA primers are replaced by DNA through exonuclease activity of DNA polymerase I
• Gaps are filled and nicks are sealed by DNA ligase

Termination

• After chromosome replication, termination must occur
• Bacterial topoisomerase IV introduces double-stranded breaks to separate interlocked circular chromosomes

Gyrase and Topoisomerase IV

• Bacterial DNA gyrase and topoisomerase IV are distinct from their eukaryotic counterparts
• These enzymes are targets for quinolones

Pre-replication Complex

• Forms at the origin of replication, including helicase and other enzymes
• Involves topoisomerase, single-stranded binding protein, RNA primase, and DNA polymerase

Nucleotide Addition

• Eukaryotic DNA polymerase adds nucleotides only in the 5' to 3' direction
• In the leading strand, synthesis continues until the chromosome end is reached

Lagging Strand Synthesis

• DNA is synthesized in short stretches initiated by a separate primer
• The linear chromosome end lacks a place to make a primer for the final fragment

Telomeres

• The ends of linear chromosomes
• Consist of noncoding repetitive sequences
• Protect coding sequences from being lost

Rolling Circle Replication

• Begins with enzymatic nicking of a double-stranded circular molecule at the double-stranded origin (dso) site
• DNA polymerase III binds to the 3'-OH group of the nicked strand, beginning unidirectional replication

Un-nicked Strand

• Replicates displacing the nicked strand
• RNA primase synthesizes a primer to initiate DNA replication
• At the single-stranded origin (sso) site of the single-stranded DNA (ssDNA) molecule

RNA Transcription

• Requires partial unwinding of the DNA double helix, forming a transcription bubble
• Transcription proceeds from one DNA strand, the antisense strand which acts as a template

RNA Product

• Complementary to the template strand
• The sense strand is almost identical to the non template DNA strand

RNA Polymerase

• RNA Polymerase adds nucleotides one by one to the 3'-OH group, similar to DNA polymerase
• However, RNA polymerase does not require a primer

Ribonucleotide

• During transcription, it is complementary to the DNA template strand
• A covalent phosphodiester bond is formed creating the growing RNA strand

E. coli RNA Polymerase

• Comprises six polypeptide subunits, five of which form the core enzyme
• Responsible for adding RNA nucleotides

Sigma Factor

• Enables RNA polymerase to bind to a specific promoter
• Allows transcription of various genes
• Various σ factors allow for transcription to different genes

Initiation of Transcription

• Transcription begins at a promoter, a DNA sequence where transcription machinery binds
• The nucleotide pair in DNA double helix corresponding to where the first 5' RNA nucleotide is transcribed is the initiation site

Elongation Phase

• σ subunit dissociates
• The core enzyme synthesizes RNA complementary to the DNA template in a 5' to 3' direction
• Rate of approximately 40 nucleotides per second

Process

• As elongation proceeds, DNA is continuously unwound ahead of the core enzyme
• It is then rewound behind it

Termination of Transcription

• Bacterial polymerase dissociates from the DNA template, releasing the newly made RNA
• The DNA template contains repeated nucleotide sequences that act as termination signals

Bacterial Polymerase

• Stalls and releases from DNA template
• This frees the RNA transcript

Eukaryotic mRNAs

• Monocistronic, encoding a single polypeptide each
• Prokaryotic mRNAs (bacteria and archaea) are commonly polycistronic, encoding multiple polypeptides

Protein-Encoding Primary Transcripts

• Must undergo processing (several steps to avoid degradation) to protect the molecules as they are transferred from the nucleus to the cytoplasm

Processing Steps

• A special 7-methylguanosine nucleotide, called the 5' cap, is added to the 5' end of the growing transcript
• After elongation, approximately 200 adenine nucleotides, called the poly-A tail, are added to the 3' end

RNA Splicing

• Process of removing intron-encoded RNA sequences and reconnecting those encoded by exons
• Facilitated by spliceosomes containing small nuclear ribonucleoproteins

Genetic Code

• The three-nucleotide code has 64 possible combinations
• There are more codons than amino acids, yielding redundancy known as degeneracy

Codon Positions

• The first two positions in a codon are critical
• The third position, called the wobble position, is less critical
• Sometimes can be changed without changing the amino acid coded for

Stop/Nonsense Codons

• 3/64 triplets which do not code for an amino acid, instead terminating protein synthesis, releasing the polypeptide from the translation machinery.

AUG

• AUG can serve as a start codon
• It also has a function for specifying the amino acid methionine
• The reading frame is set by the AUG start codon

mRNA Translation

• Each set of three nucleotides following the start codon is a codon in the mRNA message
• Molecule can be simultaneously translated by many ribosomes, all synthesizing protein in the same direction

Reading

• The mRNA is read from 5' to 3' and polypeptide is synthesized from N terminus to C terminus
• The complete structure of mRNA with related ribosomes is called a polyribosome or polysome

Bacteria and Archaea

• Before transcriptional termination, each protein-encoding transcript begins synthesis of many copies of the polypeptide
• Transcription and translation concurrently form polyribosomes because both happen in the 5' to 3' direction
• Both occur in the cytoplasm and the RNA transcript is not processed

tRNA Structure

• Mature tRNAs take on a three-dimensional structure when complementary bases expose RNA bonding
• This shapes an amino-acid binding site
• Called the CCA amino acid binding end
• The anticodon is at the other end

tRNA's Bases

• Cytosine-cytosine-adenine sequence at the 3' end of the tRNA
• The anticodon at the other end is a three-nucleotide sequence that bonds mRNA through complementary base pairing

Amino Acid Addition

• An amino acid is added to the end of a tRNA molecule in tRNA charging
• Each tRNA is hooked to its precise cognate amino acid with enzymes called aminoacyl tRNA synthetases

Process in tRNA

• At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids
• The amino acid is first activated with adenosine monophosphate and transferred to the tRNA forming a charged tRNA
• Afterwards, AMP is released

Protein Synthesis Initiation

• Begins with forming an initiation complex
• In E. coli, the complex contains the small 30S ribosome, the mRNA template and three initiation factors
• Guanosine triphosphate (GTP) is the energy source
• An initiator tRNA carries N-formyl-methionine (fMet-tRNAfMet)

tRNA Interaction

• The initiator tRNA interacts with the start codon AUG, carrying a formylated methionine (fMet)
• fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli
• It is involved in the initiation

Shine-Dalgarno Sequence

• In E. coli mRNA, the leader upstream of the first AUG codon is called the Shine-Dalgarno sequence interacting with ribosomal rRNA

Location Anchoring

• Anchors 30S ribosomal subunit on the mRNA template

Ribosome Binding

• At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome

Eukaryotes

• They differ from the bacteria
• The initiator tRNA is a different tRNA carrying methionine called Met-tRNAi
• The eukaryotic initiation complex recognizes the 5' cap rather than the Shine-Dalgarno sequence

Eukaryotic Strand

• The eukaryotic initation complex tracks along the mRNA in the 5' to 3' direction until the AUG start codon is recognized
• At the start codon is the complex of Met-tRNAi,mRNA and the 40S subunit

Protein Synthesis Elongation

• Bases of translation are the same in prokaryotes and eukaryotes
• In E. coli the binding of the 50s ribosomal subunit to produce makes ribosomal sites

Three Funcional Ribosomal Sites

• The A (aminoacyl) site binds incoming charged aminoacyl tRNAs
• The P (peptidyl) site binds tRNAs carrying amino acids bonded with polypeptide chains
• The E (exit) site releases discharged tRNAs that can be recharged with free amino acids

Assembly Line

• During initiation bacterial or eukaryotic tRNA enters the P site
• A offers a free site to accept the tRNA corresponding to the first codon after the AUG

Peptide Bond

• Formation is catalyzed by peptidyl transferase
• It is an RNA-based ribozyme integrated into the 50S ribosomal subunit

tRNA Link

• The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain

Translation Termination

• Occurs when a nonsense codon (UAA, UAG, or UGA) is encountered
• Nonsense codons are identified by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA

Process of Breaking Strands

• The small and large ribosomal subunits dissociate from the mRNA and from each other
• They are recruited almost immediately into another translation ination Complex

Mutations

• Silent mutation: no effect on protein structure
• A missense mutation results in a different amino acid
• Conditional mutations show effects only under certain conditions

Point and Nonsense Mutations

• Converts a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon)
• Frameshift mutations caused by insertions or deletions are very bad

Chemicals

• Nucleoside analogs are like bases
• They are often incorporated into DNA during replication

Base Analogs

• Cause mutations because they often will have different paring rules than the bases
• Nitrous acid deaminates cytosine which is converted to uracil
• Adenine is deaminated to hypoxanthine resulting in the conversion of TA to CG

Chemical Mutagens

• Intercalating agents work differently at the stacked nitrogenous levels
• Leads to a shift depending on a skip/insertion

Ionizing/Non-Ionizing Radiation

• Causes a mutation in DNA
• Ionizing radiation has very strong radiations that break down DNA
• UV light is not energetic so it uses chemical changes

Formation

• Non-ionizing radiation can induce dimer formation
• The most common base is thymines with the strand
• Both replication and transcription are stopped and unrepaired because polymerase has difficulty

Bacteria

• Bacteria uses 2 mechanisms for repairing Thymine Dimers
• Nucleotide Excision Repair and Photoreactivation

Plating

• Replica Plating is used to identify bacterial mutants
• Used to detect nutritional mutants

The AMES Test

• Devised by Bruce AMES in 1970s
• Method that quickly determines toxins with bacteria

Vertical Gene Transfer

• The transmission of genetic information throughout generations via reproduction - the main mode of transferring
• Horizontal Gene Transfer, the intro of genetics within 1 generation

Horizontal Gene Transfer

• The intro of genetics within 1 generation
• Genes share, influence their phenotypes, and is more common in prokaryotes

Transformation

• Prokaryotes take up the DNA
• Binds and Transported and made single stranded

Antimicrobial Resistance

• Genes encode resistance in compounds
• Can be viruses
• Can be transferred through conjugation

Conjugation

• Conjugation is directly through a conjugation plus and organisms
• The F plasmid
• Plus: Donor
• Negative: Recipient

F Plasmid

• Bacteria chromones
• The integration with the plasmid

R Plasmids

• Encode proteins
• Transfer of cells for the species