U6 mlc genetics megaset

Nucleic Acids

M: nucleotides

P: DNA and RNA

B: phosphodiester bonds

CHONP

Nucleic Acid Functions

• information

• protein synthesis

• regulation

• energy

• structure

Nucleotide

• nitrogen base

• 5 carbon sugar

• phosphate group (1-3)

know how to number sugar carbons

Deoxyribose vs Ribose

Ribose has two OH C-2’ and C-3’

Deoxyribose only OH at C-3’

Nitrogen bases

pyrimidines- single ring (C, T, U)

purines- double ring (A, G)

• purines can hydrogen bond to pyrimidines

• C-G - 3 H bonds

• A-T,U - 2 H bonds

Nucleoside

nitrogen base + sugar

Polymer structure of Nucleic Acids

• Outside: Phosphate backbone

• Inside: Complementary nitrogen bases

• Double Helix

• Each strand has a 5’ and a 3’ end

  • Run in opposite direction (antiparallel)

  • New nucleotide are added to 3’ end

    • Synthesized 5’ → 3’ direction!

    • “We” write 5’ → 3’ direction

    • “Cells” read 3’ → 5’ direction

Prokaryote vs Eukaryote Genetic Code

• Eukaryotes

  • Linear chromosomes

  • Nucleus (membrane bound)

• Prokaryotes

  • Circular

  • Nucleoid (not membrane bound)

DNA vs RNA (Eukaryotes)

see img

Central Dogma of Genetics

DNA → mRNA→ Protein

Transcription: DNA → mRNA

Translation: mRNA → Protein

RNA breaks down easily → signal is easily regulated

BPQ What is one reason that mRNA made of RNA rather than DNA

Hint: mRNA is a messenger (a signal)

DNA Synthesis vs Transcrip. vs Transla.

Synthesis: DNA → DNA

Transcription: DNA → mRNA

Translation: mRNA → Protein

for cell division

S phase

BPQ

Why does DNA synthesis occur?

In Eukaryotes, when does DNA synthesis occur?

Semi-conservative nature of replication

• DNA composed of 2 strands

  • Each strand provides a template to create a new strand

  • Each duplicated DNA consists of 2 strands:

    • 1 old strand (template strand)

    • 1 new strand

Replication Steps

1. Unwind

2. Unzip

3. Prime

4. Copy

5. Replace

6. Conjoin

All occurs in close proximity to replication fork

Unwind

Topoisomerase- relaxes DNA supercoiling in front of the replication fork

Unzip

Helicase- breaks hydrogen bonds between two template strands

• Single-stranded DNA binding protein

• Binds to DNA and prevents reannealing

Prime

DNA replication needs a starting place

Primase- adds an RNA primer from which replication can start rfom

Copy

DNA Polymerase III- creates new DNA strand that is complementary to the template strand

• Starts from RNA primer

• synthesizes DNA in 5’→3’ direction ONLY

the new strands are made in opposite directions

BPQ

DNA strands are antiparallel, so how will DNA replication look differently for each strand?

Leading and Lagging Strand

bc DNA must be made 5’→3’

• Leading Strand

  • Grows in the 5’ → 3’ direction

  • Grows continuously

• Lagging Strand

  • Grows in the 3’ → 5’ direction

  • Grows discontinuously in 5’ → 3’ chunks

    • Okazaki fragments

Replace

DNA Polymerase I- replaces RNA primers with DNA

Conjoin

DNA Ligase- connects disjointed ends of DNA backbone in the new strands

e.g. between Okazaki Fragments

Proofreading

DNA Polymerase II

• Moves slower than DNA polymerase III

• Makes less mistakes than DNA polymerase III

• Used for proofreading

DNA replication in Prokaryotes vs Eukaryotes

• Prokaryote

  • Chromosomes are circular

  • Chromosomes are smaller

  • 2 replication forks

  • 1 replication bubble

• Eukaryotes

  • Chromosomes are linear

  • Chromosomes are larger

  • Many replication forks

  • Many replication bubbles

Replisome

• The molecular machinery at the replication fork working in unison

  • Helicase, polymerase, ligase, etc.

Plasmids

Small segments of circular DNA that are separate from the chromosomes and replicate independently

2 major Prokaryote groups

• archaebacteria

• eubacteria

Central dogma of genetics locations

• DNA → mRNA → Protein

• Transcription: DNA → mRNA

  • Nucleus

• Translation: mRNA → Protein

  • Ribosomes (ER or cytoplasm)

RNA

• All RNA is created in the nucleus

• Messenger RNA (mRNA)

  • Carries the genetic signal from the nucleus to the ribosomes

• Ribosomal RNA (rRNA)

  • Makes a large part of the physical structure of the ribosome

• Transfer RNA (tRNA)

  • Pulls amino acids into the ribosome to by synthesized as prescribed by the mRNA

• Micro RNA (microRNA)

  • Regulates mRNA translation and breakdown

    • Post-transcriptional regulation

Transcription

• DNA → mRNA

• Steps

  • Initiation

  • Elongation

  • Termination

  • Post-transcriptional* modifications

RNA Polymerase

The multipurpose enzyme that makes pre-mRNA

• Multipurpose

  • DNA synthesis

    • Unwind – Topoisomerase

    • Unzip – Helicase

    • Prime - Primase

    • Copy – DNA Polymerase III

    • Replace – DNA Polymerase I

    • Conjoin – Ligase

  • Transcription

    • Unwind – not needed

    • Unzip – RNA Polymerase

    • Prime – RNA Polymerase

    • Copy – RNA Polymerase

    • Replace – not needed

    • Conjoin – not needed

in transcription, only opening small segments of DNA → won’t supercoil

BPQ

Why does transcription not need a topoisomerase to unwind the DNA?

Initiation

• RNA polymerase binds to a sequence of DNA called the promoter

  • Promotor contains recognition sites called TATA boxes in Eukaryotes and Archaebacteria

• Separates (unzipped) the DNA

  • Template strand (3’ → 5’)

  • Coding strand (5’ → 3’)

Elongation

RNA Polymerase reads the template strand (3’ to 5’) and creates pre-mRNA in the 5’ → 3’ direction.

Termination

• RNA Polymerase transcribes the terminator signal that causes pre-RNA to separate from the template strand and RNA polymerase

  • In eukaryotes this terminator transcribes a hairpin loop

    • Called Rho independent termination

2 types: Rho independent and Rho dependent termination

Rho Independent Termination

• Used by Eukaryotes and some prokaryotes

• Transcription is terminated by a hairpin loop in the mRNA

Rho Dependent Termination

• Used by some prokaryotes

• Transcription is terminated by Rho factor

complementary bases will create a hairpin loop (Rho indep. term.)

BPQ

What kind of pattern in the genetic code would create a hairpin loop?

Post-transcriptional Modification

• 5’ cap and 3’ poly-A tail

  • Helps protect the mRNA for exonucleases in the cytoplasm

  • Eukaryotes only

• RNA splicing

  • Removes introns

  • Pre-mRNA → mature-mRNA

  • Eukaryotes and Archaebacteria only

mRNA metabolism

• RNA is less stable than DNA

  • mRNA is given a 5’ cap and a 3’ poly A tail to protect it.

• Cytoplasm is full of de-capping proteins and exonuclease

  • Exonucleases – enzyme that removes nucleic acids one at a time

  • mRNA is intentionally broken down

  • mRNA can have a half life of a few minutes

RNA Splicing (introns and exons)

• Not all the RNA transcribed will be apart of mRNA that leaves the nucleus

  • Exons --  become part of the final mRNA

    • Exons “exit” the nucleus

  • Introns -- are removed

Alternate Splicing

a cellular process in which exons from the same gene are joined in different combinations, leading to different, but related, mRNA transcript and different proteins w/ different structures/functions

One region of DNA can code for multiple proteins by using different exons

Translation

• Process of polypeptide chain from mRNA sequence at a ribosome

  • Prokaryotes

    • Occurs simultaneously with transcription

    • No post-transcriptional modifications (in Eubacteria)

  • Eukaryotes

    • Must exit the nucleus first

    • Post-transcriptional modifications

• Notice how multiple ribosomes can translate the same mRNA

mRNA doesnt travel far in prokaryotes bc prokary. cells are smaller

also transcrip. and transla. occur simultaneously

BPQ

What are some reason that prokaryotes don’t need a 5’ cap or poly-A tail on their mRNAs?

Codon

• 4 nucleotides / 20 amino acids

• Codon – 3 nucleotides

  • Start Codon (Methionine)

  • 20 amino acids

  • Stop Codon

Ribosome

• Large subunit

• Small Unit

• APE sites – sites for tRNA

  • A- Aminoacyl

  • P- Peptidyl

  • E- Exit

Transfer RNA (tRNA)

• “transfer” amino acids onto the growing polypeptide

• Parts

  • Anticodon

    • 3 nucleotides that are complementary to a codon**

  • D loop (do not memorize)

  • T loop (do not memorize)

  • Amino Acid attachment site (3’ end)

    • Only the amino acid that matches the codon may bind here

Wobble Pairing

Sometimes the last nucleotide in an anticodon doesn’t have to match the codon

Translation steps

1. Initiation – Ribosome assembles around the start codon

2. Elongation – Amino acids are added to the polypeptide chain

3. Termination – Release factor binds to the stop codon

Initiation steps (Prokaryotes)

1. Small subunit and first tRNA assemble (called the initiation complex)

2. initiation complex assembles around the start codon with a Shine-Dalgarno sequence before it

3. Large subunit assembles on top of the first tRNA in the P site

Initiation steps (Eukaryotes)

1. Small subunit and first tRNA assemble (called the initiation complex)

2. Initiation complex attaches to the 5’ cap of the mRNA and moves in the 3’ direction until it reaches the first start codon

3. Large subunit assembles on top of the first tRNA in the P site

Elongation

1. New tRNA enters the A site

2. Peptide bond forms between the amino acids in the A and P site

3. tRNA in the P site releases its amino acid

4. Ribosome shifts over one codon

5. tRNA that is now in the E site leaves

6. Repeat

Termination Process

• Stop codons are the only codons that do not have a tRNA that are complementary to  them

  • The are recognized by a protein called release factor

1. Ribosome shifts and there is now a stop codon in the A site

2. Release factor (a protein) enters the A site and binds to the stop codon

3. Ribosome complex completely breaks apart releasing the polypeptide

phosphorylated, chaperone proteins help fold it, etc.

a ton of things can happen before a polypeptide becomes a protein

Review Question

What can happen to a polypeptide after it is translated?

modRNA (Nucleoside-modified messenger RNA)

• Synthetic mRNA made with nucleotide analogues

  • Nucleotides are not A,U,C, or G

  • But are read as A,U,C, or G by the ribosome

• Used to trick the cell into coding for a specific protein

  • Moderna and Pfizer COVID-19 vaccine

• Why use nucleotide analogues over natural nucleotides?

tba; prob make it easier to get into cell

Review Question

The modRNA in the COVID-19 vaccines are put into microscopic lipid nanoparticle, why?

Initiation complex

The complex of the Small ribosome subunit + first tRNA assembling in initiation (translation)

turn genes on/off

BPQ

Why regulate gene expression?

multicellular has cell specialization → altho every cell is genetically the same, diff genes expressed in diff cells

BPQ

How might regulation in be different in multicellular organism than unicellular organism?

turning genes on/off leads to evolution (changes over time)

BPQ

How might gene expression effect heredity and evolution?

Factors that can affect gene expression

• Transcriptional (Prokaryotes & Eukaryotes)

  • Transcription factors (Pro & Eu)

    • Activators

    • Inhibitors/Repressors

  • Histones (Eu & Archae)

  • DNA Methylation (Pro & Eu)

• Post Transcriptional (Eu)

  • MicroRNA - miRNA

  • Alternate splicing

Transcription

Promoter – region where RNA polymerase binds

RNA polymerase creates mRNA from the template strand

Regulatory Sequence

• a noncoding region of DNA that can influence gene expression.

  • Promoter (Pro & Eu)

  • Operator (Pro)

  • Enhancer (Pro & Eu)

  • Silencers (Eu)

Regulatory Gene

a coding gene that produces a product that can influence gene expression

Transcription Factor

a protein that can influence gene expression by binding regulatory sequences.

Activator – turns on / promotes transcription

Repressor – turns off / decreases transcription

Prokaryotic Operon

• a series of related genes where transcription is initiated by one promoter

  • Creates 1 mRNA & multiple proteins

• often regulated by a repressor that attaches downstream of the promoter in a sequence called an operator (a regulatory sequence)

• Repressor physically blocks RNA polymerase

all the genes in an operon are LINKED

1 gene per promoter allows for complex expression in eukaryotes

BPQ

In Eukaryotes 1 promoter always transcribes 1 gene and only 1 gene.  What might be some reasons for this?  Why do Eukaryotes not use operons?

Lac Operon (Repressor: Inducible)

• Inducible Operator

• Lactose is an Inducer that triggers the transcription of genes responsible for the breakdown of lactose

Inducible Operator

transcription is “turned off” until an inducer binds to the repressor

• Inducer releases the repressor from the operator

Lac Operon (Activator)

• Activators bind to enhancers (regulatory sequence) and guide RNA polymerase onto the promoter.

• cAMP is a coactivator that is produced in the absence of glucose.  cAMP “turns on” the activator CAP and triggers transcription of genes responsible for the breakdown of lactose

1. off

2. off

3. off

4. on

both must be “on” for Lac Operon to turn on

BPQ

Determine if the Lac Operon is turned “on” or “off”

1. Glucose present, Lactose present

2. Glucose absent, Lactose absent

3. Glucose present, Lactose absent

4. Glucose absent, Lactose present

Trp Operon (Repressor: Repressible)

• Repressible Operator

• Tryptophan is a corepressor that stops the transcription of genes responsible for the synthesis of tryptophan

Repressible Operator

• transcription is “turned on” until a corepressor binds to the repressor.

  • Corepressor enables the repressor to bind to the operator

lactose comes from an inducible operator - off by default; lactose presence turns on the lactase gene

tryptophan comes from a repressible operator - on by default; trp presence turns off the gene bc there’s enough trp now

BPQ

Why does Lactose “turn on” gene expression while Tryptophan “turn off” gene expression?

Eukaryote Transcription Factors

• Transcription factors

  • Activators

  • Inhibitors

  • Basal/general transcription factors

• Regulatory sequences

  • Enhancer (upstream of the promoter)

  • Silencer(upstream of the promoter)

  • Promoter

• **unlike the Prokaryotes with their operators, Eukaryotes have no regulatory sequences downstream of the promotor

Basal/General Transcription Factors

• All Eukaryotic genes require basal transcription factors for the RNA polymerase to bind to the promoter

  • RNA Polymerase in prokaryotic cells can often bind on its own

Upstream Regulatory Sequences

• Eukaryotic regulator sequences can be very far upstream from the promotor

  • 1 regulator sequence can affect multiple promotors/genes

• Prokaryotic regulator sequences are always proximal to the promotor

  • Can only effect 1 promotor(but multiple gene/see operon)

DNA Methylation

• A methyl group can be added to the nitrogen bases C or A (usually C)

  • Affect promotors

    • “Turn off” gene expression

  • Affect Histones

    • “Turn off or on” gene expression

• often leads to more long-term patterns in gene expression

growth hormones for cell specialization

BPQ

Considering the long-term effects of DNA methylation, what is likely a common application of DNA methylation in multicellular organisms?

Post Transcriptions Gene Expression (Eu)

• MicroRNAs (miRNA)

  • Small noncoding RNAs that can bind to mRNA

    • Prevent translation

    • Accelerate mRNA breakdown

• Alternate splicing

Gene Expression and Cell Differentiation

Multicellular Eukaryotes need to express different genes in different cells

see which genes are turned on/off due to cell specialization (?)

BPQ

A Liver tumor is biopsied and found to be metastatic lung cancer

How can we tell that a tumor in the liver came from the lungs?

Gene Expression and Evolution

Changes in gene expression are often more consequential than changes to the genes themselves

Mutation

• change in DNA sequence

• good, bad, neutral

mutations in regulatory sequences (e.g. promoters) (affect how MUCH of a protein is made)

BPQ

Can a mutation affect a phenotype w/o affecting coding (transcribed and translated) DNA? how?

Mutation Effects

1. Nothing - non-coding DNA (junk DNA), introns, 3rd nucleotide in codon

2. AA seq. - coding DNA

3. Protein Output - Reg. seq. & reg. genes

Mutations in Coding DNA

1. Missense Mutation

2. Silent Mutation

3. Nonsense

4. Insertion/deletion

   1. Frameshift

Missense mutation

change 1 nucleotide (any of 3 in codon) to another resulting in AA change

Silent mutation

change 1 nucleotide to another BUT does NOT result in AA change (3rd nucleotide sometimes)

no effect sometimes

Nonsense Mutation

changes AA to stop codon

Insertion/deletion mutation

addition/subtraction of nucleotide

If 3, 6, 9, etc. nucleotides are added, a frameshift mutation does NOT occur

Frameshift Mutation

changes all AA downstream when the insertion/deletion is not divisible by 3

3rd nucleotide b/c most often causes silent mutations that tend to have no effect

BPQ

In a coding gene found across multiple species, where would we most likely find differences in nucleotides?

• First nucleotide of a codon

• Second nucleotide of a codon

• Third nucleotide of a codon

Chromosomes (Human vs Chimpanzee)

Human – 46 Chromosomes (22 autosomes, 2 sex chromosomes)

Chimpanzee  – 48 Chromosomes (23 autosomes, 2 sex chromosomes)

• in chromosome 2, either humans put the two chimp chromosomes tgt, or chimps split them apart after we diverged

• but overall the genes are the same, the chromosomes in 2 are just split (shows we are closely related!)

Karyotype

image of all the chromosomes in a single cell

M phase (?)

Review Question

What stage of the cell cycle does the karyotype show

Extra/Missing Chromosomes

1. Euploid

2. Aneuploid

3. Polyploidy

Euploid

having correct number of chromosomes (humans = 46)

Aneuploid

addition or loss of a chromosomes (1 pair)

1. Monosomy - missing a chromosome from a pair

2. Trisomy - having an extra chromosome in a pair