DNA replication

Initiation of DNA replication

• Helicase binds to the origin of replication

• Helicase begins to unzip the double helix by breaking the hydrogen bonds between the bases

• This creates a replication fork - the region where the original DNA double helix splits into two strands

• Single-binding proteins bind to the single-stranded DNA to keep the strands separate by preventing the hydrogen bonds from reforming

• The process of unzipping the double helix creates supercoils and tension ahead of the replication fork that could damage the DNA

• Gyrase/topoisomerase moves in front of helicase to relieve the tension

Synthesizing the Complementary Strand

• DNA polymerase III is the enzyme that will read the template and build the complementary strand

• It can only add new nucleotides to an existing strand at the 3’ end

DNA polymerase III builds the new DNA strand in the 5’ to 3’ direction

DNA polymerase III is an enzyme with an active site that is specifically shaped. Which is why DNA polymerase III can only build the new strand in the 5’ to 3’ direction.

• Primase creates an existing strand for DNA pol III to add to the 3’ end

• This is accomplished by adding a primer made of a short sequence of RNA nucleotides

• Once the primer is laid down, DNA pol III can attach to the new strand and begin synthesizing the complementary strand to the template

DNA proofreading

• DNA pol III can proofread the newly formed DNA strand as it is being built

• If a mistake is made, the mismatched base will be removed and replaced with the correct one

DNA pol III is not 100% accurate with the DNA replication process and proofreading.

Mistakes are still (rarely) made!

These “mistakes” are mutations and create variations for natural selection to act upon

Removing primers

• Remember: The primers are made of RNA nucleotides (ribose sugars and uracils)

• DNA Polymerase I removes the RNA nucleotides and replaces them with the correct DNA nucleotides

The replication fork has directionality

• Remember: The strands of DNA are antiparallel

• Which helicase unzips the double helix, one strand runs 5’ to 3’ and the other strand runs 3’ to 5’

DNA replication proceeds differently on the two strands because they are antiparallel

Leading v. Lagging Strand

• DNA pol III can synthesize one of the strands continuously following the same direction as helicase - this is the leading strand

• The other strand is synthesized discontinuously AWAY from the replication fork - this is the lagging strand

Replication of the leading strand

• Only 1 primer is required to start replication on the leading strand

• Once the primer is created, DNA pol III follows the direction of helicase until the whole molecule has been unzipped

Replication of the lagging strand

• The lagging strand is replicated in sections with DNA pol III moving away from the replication fork

• These fragments are called Okazaki fragments

• Each fragment needs its own primer

Protein synthesis: Transcription

RNA structure review

• Sugar: Ribose

• Bases: A, G, C, and Uracil

• RNA is typically a single polynucleotide strand that can then (but not always) be folded into a 3D shape

Protein synthesis

The Central Dogma

• The central dogma describes the flow of genetic information

• DNA to RNA to protein

• DNA is transcribed into mRNA

• mRNA is translated into a polypeptide

Transcription

Transcription

• Producing mRNA using DNA as a template

• Occurs in the nucleus of eukaryotic cells

• Occurs in the cytoplasm of prokaryotic cells

Why is transcription necessary?

Allows for:

• Only a portion of the genome to be copied - resource efficiency

• DNA to remain protected in the nucleus of eukaryotes

RNA polymerase

• Performs transcription (elongating the mRNA strand) sing the DNA template strand as a guide

• RNA polymerase must synthesize the mRNA strand in the 5’ to 3’ direction

Phases of Transcription

1. Initiation

2. Elongation

3. Termination

Transcription Initiation

• A promoter is a non-coding region of DNA in front of the gene of interest that begins with the TATA box

• Transcription factors are proteins that bind to the promoter

• Transcription factors then recruit RNA Polymerase to the promoter

• RNA polymerase begins to temporarily unzip a small section of the double helix to expose the bases

Transcription elongation

• RNA polymerase “reads” the template strand (antisense strand) to6 synthesize the mRNA

• Remember: The mRNA is synthesized in a 5’ to 3"‘ direction

• As the mRNA is synthesized, the RNA nucleotides will temporarily hydrogen bond with the template strand

• The growing mRNA strand exits RNA polymerase and the DNA re-zips

Template Strand (antisense strand) - the strand of DNA that RNA polymerase “reads”

Coding strand (sense strand) - The complementary DNA strand to the template strand

Transcription termination

• A terminator sequence at the end of the gene is reached

• Signals for RNA polymerase to release the mRNA and detach from the DNA

• Transcription is now finished

Regulation of transcription

• Non-coding regions of DNA are sections of the DNA that don’t code for a protein

• Some parts help with regulation

• Enhancers - increase rate of transcription

• Silencers - Decrease rate of transcription

Other non-coding regions of DNA

• Telomeres -

  • Repetitive sequences at the end of linear (eukaryotic) chromosomes

  • Protect the ends of the chromosomes

• Genes for rRNA and tRNA

  • RNA is synthesized from these genes, but they don’t code for proteins

  • Create the other 2 RNA types

• Introns

  • Base sequences hat get removed from the mRNA after transcription

  • Only in eukaryotes

Post-transcriptional Modification (mRNA processing)

mRNA processing

• Done in eukaryotic cells after transcription and before the mRNA can leave the nucleus

• Converts pre-mRNA into mature mRNA

• Includes:

  • mRNA splicing

  • Addition of the 5’ cap and poly-A tail

5’ Cap and Poly-A tail

• 5’ cap is a modified nucleotide that is added to the 5’ end of the mRNA

• Poly-A tail is a string of adenines attaches to the 3’ end of the mRNA

• The 5’ cap will help with ribosome binding during translation

• Both aid in the export of the mature mRNA from the nucleus and protect the mRNA from degradation in the cytoplasm

mRNA Splicing

• Pre-mRNA contains:

  • Exons -base sequences that are expressed (aka coding regions within a gene)

  • Introns - base sequences that are removed before translation

Introns are removed from the mRNA during mRNA splicing (introns stay in the nucleus)

• snRNPs are small Nuclear Ribonucleoproteins that catalyze splicing

• snRNPs bind to either side of the introns and then assemble into spliceosomes

• Splicesomes remove the introns and ligate the exons together

Alternative splicing

• Different introns are removed - this creates unique polypeptides

• One gene (and thus one pre-mRNA) can provide instructions for several different polypeptides due to alternative splicing

Protein synthesis: Translation and Mutations

Protein Basics

• Elements: CHON

• Monomer: Amino acid

  • 20 different AAs

  • 20 different side chains

• Polymer: Polypeptide

• Covalent bond: Peptide bond

4 Levels of protein structure

• 1”- polypeptide chain

• 2” - alpha helices and beta pleated sheets

• 3” - 3D structure determined by side chains

• 4” - 2+ polypeptide chains interacting

• The structure determines the function

Protein synthesis

Review: The central Dogma

• DNA to RNA to Protein

• Transcription: DNA to mRNA

• Translation: mRNA to Protein

Protein Synthesis in Prokaryotes

• In Prokaryotes, translation can occur immediately after transcription

• This causes protein synthesis to be faster in prokaryotes compared to eukaryotes

Protein synthesis in Eukaryotes

• Transcription occurs in the nucleus

• mRNA processing occurs before the mature mRNA can leave the nucleus

• Translation occurs in the cytoplasm either by:

  • Free ribosomes

  • Attached are ribosomes (on the rough ER)

The genetic code

• How mRNA is “decoded” into amino acids

• mRNA is “read” in triplets of bases called codons

• each codon codes for a specific amino acid

Circle Codon wheel

How to use:

1. Start in the very middle circle to find the first base in the codon

2. Follow that pie slice out to the next circle and find the second base in the codon

3. Follow that pie slice out to the third base in the codon

4. The amino acid is listed outside the outer ring of bases

Rectangle codon chart

how to use:

1. On the left-hand side, find the first base of the codon

2. Across the top, find the second base of the codon

3. On the right-hand side, find the third base of the codon

4. The amino acid will be listed in the cell that is intersected by all of those columns/rows

4 special codons

• Start codon: AUG - this is where translation will always start

• Stop codons: UGA, UAA, UAG - when one of these three codons is “read”: translation will end

Characteristics of the Genetic Code

• Universal

  • Nearly every organism on earth uses the same genetic code

  • “Ver few exceptions”

  • Evidence of LUCA

  • Basis of several biotechnology techniques

• Redundant/Degenerate

  • Some amino acids are coded for by more than one codon

  • Example:

    • GGU, GGC, GGA, GGG

      • These are all codes for Glycine (Gly)

• Unambiguous

  • No codon specifies more than one amino acid

Implication of the universal genetic code

• Allows for genetically modified organisms

• Splice the gene(s) of interest into another organism, allows for protein synthesis of the gene(s) of interest by the new organism

Uses of Genetically modified organisms

• Insulin gene in bacteria - allows for mass production of the insulin protein

• Pesticide gene in crops- allows for the crops to produce a protein that acts as a pesticide so bugs don’t eat the crop

During translation, the mRNA is read by the ribosome and using the code within the mRNA, amino acids are added in a specific sequence to form a polypeptide

3 types of RNA

• mRNA = messenger RNA (take the message from DNA and brings it to the ribosomes)

• tRNA = transfer RNA (carries amino acids to the ribosomes)

• rRNA = ribosomal RNA (structural component of ribosomes)

tRNA structure

• Single strand of RNA that is folded into a 3D structure

• 2D representation looks like a clover

• The 3D structure is held together by hydrogen bonds between complementary base pairs

• The bottom of the tRNA contains the anticodon (a group of 3 bases in the tRNA that will bind to the mRNA codon)

• The top of the tRNA is the Amino Acid Binding Site (where the amino acid gets attached)

tRNA function

1. The enzyme aminoacyl-tRNA synthase attaches the correct amino acid to the tRNA

2. the tRNA brings the amino acid to the ribosome and binds to the mRNA codon

Ribosome structure

• Made of rRNA and proteins

• Made of 2 subunits:

  • Small subunit

  • Large subunit

• mRNA attaches to the small subunit

• Large subunit has 3 binding sites for tRNA (A, P, and E sites)

APE Binding sites

• A site = Aminoacyl-tRNA binding site (for the incoming tRNA with the next amino acid)

• P site = Peptidyl-tRNA binding site (for the tRNA “holding” the growing polypeptide chain)

• E site = Exit site (for the discharged/”empty” tRNA to leave the ribosome

Ribosomes facilitate:

1st - the binding of the mRNA codon with the tRNA anticodon using complementary base pairs

2nd - the formation of a peptide bond between the incoming amino acid and the growing peptide chain

The complementary nature of the codon and anticodon ensures that the correct amino acid is placed in the polypeptide sequence

Phases of translation

1. Initiation

2. Elongation

3. Termination

Translation Initiation

• the 5’ end of the mRNA binds to the small ribosomal subunit

• The small ribosomal subunit moves from the 5’ to the 3’ end of the mRNA and “scans” for the tart codon

• At the start codon, the initiator tRNA binds to the start codon

• The large ribosomal subunit assembles, placing the initiator tRNA into the P site

Translation Elongation

• After initiation, the Ribsosome begins to “read” the mRNA in codons

• The following cycle occurs:

  • Codon Recognition

  • Peptide bond formation

  • Translocation

Codon recognition

• Incoming tRNA’s anticodon binds to the mRNA’s codon in the A site

• Remember: the codon-anticodon binding is complimentary

Codon recognition ensure that the correct amino acid is added into the polypeptide sequence

Peptide Bond formation

• Peptide bond is formed between the polypeptide chain and the new amino acid

• This transfers the chain from the tRNA in the P site to the tRNA in the A site

Translocation

• the ribosome shift over one codon

• The discharged tRNA in the P site will move to the E site and exit

• The tRNA in the A site that is now carrying the growing polypeptide chain will move into the P site

• The A site will now be empty for the next incoming tRNA

The elongation cycle will repeat until a stop codon is reached

Translation Termination

• When a stop codon is in the A site, no new tRNA will enter

• Release factor will enter and will perform a hydrolysis reaction to break the bond between the polypeptide chain and the tRNA in the P site

• After the polypeptide chain is released from the tRNA the translation complex disassembles

• The polypeptide chain will then be modified and folded into this final structure/functional form

Post-Translational Modification

Post-Translational Modifications

• Often occur in the golgi

• Can involve:

  • Addition of chemical groups (ex. Phosphates or sugars)

  • Cleavage of specific peptide bonds

Pre-Proinsulin to Proinsulin

Pre-proinsulin:

• 110 amino acids in length

• 4 sections:

  • Signal peptide

  • A chain

  • B chain

  • C-peptide

In the RER, the signal peptide is removed to create proinsulin

Proinsulin to Insulin

In the RER:

• Form disulfide bridges between the A chain and B chain

• Packaged into vesicles to move to the golgi

In the Golgi:

• Remove C-peptide to create insulin

Proteomes

Proteome

• the total of all proteins made and used by the body

• DYnamic - constantly synthesizing and hydrolyzing proteins

• Proteasome - protein complex that hydrolyzes damaged or unused proteins

Cell Differentiation/Specialization

• All cells in your body have the same DNA

• Cells have differentiated into different types (ex. muscle cells, neurons, heart cells, skin cells, etc.)

• Different genes are expressed (proteins produced) in those different cell types - one major thing that makes them different!

Mutations

Mutations

• Gene mutations are structural changes or alterations in the DNA sequence of an organism

• Some mutations have zero impact on an organism’s fitness, some mutations increase fitness (meaning they are an adaption) and some mutations decrease fitness

Types of mutations

• Substitutions (point mutations)

  • A single nucleotide is changed - single nucleotide polymorphism (SNPs)

  • Synonymous mutation/Silent mutation- the amino acid remains the same

  • Non-synonymous mutation - the amino acid is changed

• Insertions or deletions

  • 1 or more nuceotides are added or removed from the DNA sequence

  • results in a frameshift mutation because the genetic code is read in triplets, so the reading frame is altered

Mutations and Polypeptide Functionality

• Synonymous mutations do not impact polypeptide functionality because the correct amino acid is placed in the polypeptide

• Nonsynonymous mutations can impact polypeptide functionality, especially if he mutation was in a critical location

• Frameshift mutations normally have a DRASTIC impact on polypeptide functionality because all subsequent codons are impacted

Causes of mutations

• Errors in DNA replication or repair

• Exposure to mutagens

  • Mutagens are agents that cause mutations

  • Chemical mutagens (ex. nicotine, mustard gas)

  • Radiation (ex. UV light, X-rays)

  • Mutagens can increase errors in DNA replication/repair

Mutations are random

• Mutations can happen anywhere in the genome

• Location is random

Mutations are (mostly) random

• some bases are more likely to be mutated than others

• Cytosine can be spontaneously converted into uracil which causes issues in DNA replication if not repaired

• Remember: Mutations create variation for Natural selection to act upon

Consequences of Mutations - cell line

• Somatic cells

  • All the cells in the body except for germ cells (cells that produce sperm/eggs)

  • Mutations in somatic cells can cause issues during a person’s life, but they do not get passed on to their offspring

• Germ cells

  • Cells that produce sperm and eggs

  • Mutations in germ cells can be passed on to an individual offspring (are inherited)

Mutations and Fitness

• Neutral/silent mutations

  • Mutations that don’t affect an organism

  • Doesn’t change fitness

  • Ex. synonymous mutations (silent), or mutations in non-coding regions (neutral)

• Harmful mutations

  • Mutations that negatively affect an organism

  • Reduces fitness

  • Typically selected against during natural selection

  • Ex. Causes disease or abnormalities

• Beneficial Mutations

  • Mutations that positively affect an organism - “adaptions”

  • Increase fitness

  • Typically selected during natural selection