Molecular genetics exam 3 Need 100 baby

mRNA carries

genetic information from DNA to ribosomes

How many codons are there

• 64

• each codon has 3base pairs.

• 61 sense codons and 3 stop codons

Whats the start codon?

AUG

what are the stop codons

UAG (amber), UAA (Ochre), UGA Opal.

Polly a tail and 5’ cap help with what

stability and translation regulation on mRNA

N- terminus

is the start codon (AUG)

C terminus

Is the codon before the stop codon.

• the last codon that gets encoded

N-C NC

What was used in Nirenburgs and matthaei experiment

Polynucleotide phosphoylase, was used to make artifitial DNA without DNA templete.

• UUU = phenylalanine (Phe)

• AAA= Lysine (Lys)

• GGG= Glycine (Gly)

• CCC= Proline (pro)

Le puh got power

Features of genetic code

1. Non overlapping. One codon can not specify more than one amino acids

2. commaless, triplet codons are read in continuation, no gaps.

3. degenerate: One amino acid can be specified by more than one codon.

4. unambiguous: a single triplet specifies only one amino acid

fucking idiot what does tRNA do

It brings amino acids to the ribosome.

• each tRNA has an anticodon that is complementary to the codon mrna so it pairs nicely,

• there are 32 different types of tRNA, needed for 61 sense codons.

How do they attach? Amino acid with the tRNA

Enzyme called: aminoacyl-tRNA synthetase makes sure tRNA carries the correct amino acid. The tRNA gets charged when the enzyme attaches correct amino acid to the tRNAs 3’ end.(CCA)

• then the charged tRNA enters the ribosome, its anticodon pairs match the codons on the mRNA.

• The ribosome helps form a peptide bond between amino acids

What is a charged tRNA

A tRNA that has an amino acid attached to the 3’ end of the CCA, with the help of an enzyme called aminoacyl-tRNA synthetase.

tRNA structure

1. The T loop (TwC)
   - thymine, pseudouridine, cytosine
   - Interacts with the ribosome to help tRNA with stability during translation

2. D loop.
   - dihydrouridine
   - Recignized by aminoacyl-tRNA synthetases so that the tRNA can be charged with an amino acid

3. Variable arm:
   - Different in every tRNA can be 3-5 bp or 13-21 bp.
   -Helps with stability of tRNA during translation

4. anticodon loop. Has inosine. Helps with wobble position. WObble position is the third bp in every codon. It ensure that we dont have an abunence of tRNAs.

Advatages of wobbling in the anticodon loop

• Inosine is a modified base formed by deamination of adenosine.

• It has the ability to hydrogen bond with U C and A.

• “CGI can bind to all the codons that either end in U C or A.

• So tRNA wobbling is the reason cells dont need as many tRNAs as number of codons (61).

Ribosomes

1. cellular machines that catalyze protein synthesis

2. ribosomes are composed of ribosomal rna and proteins

Made up of the large and small subunit: all together its 80S.

• Large subunit: 60S

• Small subunit 40S

Large = 28srRNA+ 5srRNA + 5.8srRNA

Small= 18srRNA

Large subuint

APE:

A is the acceptor of the tRNA that is carrying an amino acid

P: the peptide chain that forms

E: the exit of the empty tRNA molecule.

Triple binding assay

Expirement used to find out what amino acids groups each triplet would fit.

1. Ribosome + tRNAs with amino acid + synthetic mRNA one codon

2. mix

3. In ribosome a specific tRNA with the amino acid should bind to the codon that goes with it. Unbound trnas floating around

4. filter

5. correct amino acid group found

codon biasing

multiple codons encode the same amino acid, however organisms do not use them equally. This preference is called codon bias.

Initiation for prok and euk during translation

Prok: N-formylmethionine (fmet)

EUK: Methionine regular met

Elongaion for prok and euk during translation

Prok: uses ordinary met

EUK: Uses ordinary met

Shine Dalgarno sequence

binding site on prokaryotic mRNA.

its 3-9 purine nucleotides located upstream of the initiation codon.

• Helps the ribosome know where to start translations

• Shine translate

Kozak sequence

Eukaryotes use the 5’ cap and AUG for ribosome scanning.

• helps ribosome recognize the correct AUG start codon for translation.

How ribosomes initiate translation in prokaryotes and eukaryotes

• Prokatyotes: In prok the 30S ribosome binds mRNA in such a way that AUG directly falls in the P site of the large subunit of the ribosome.

• Eukaryotes: The 40S subunit directly inds to the 5’cap and scans for AUG using the kozak sequence.

Steps in Eukaryotic translation CIET (can i eat tom)

1. Charging of tRNA, with amino acids

2. Initation: Complex formation at the Shine-Dalgarno sequence(prok) or 5’ Cap in Euk

3. Elongation: Movement of Ribosome complex on mRNA in he 5’-3’ direction, until A site reaches one of the three stop codons UGA, UAG, UAA

4. Termination: When large subunit of ribosome reaches a stop codon.

Protein factors for Prok and Euk

Initiation is IF

Elongation is EF

Termination is RF (release)

For prok there is a - sign

tRNAs for Prok vs Euk during initation and Elongation

During initation prok and Euk use different tRNAs for the AUG start codon.

• Prokaryotes use fMet-tRNA (N-formylmethionine)

• Eukaryotes use Met-tRNA (Methionine)

However, during elongation the tRNA used for prok and euk is the same, ordinary Met

• REMEMBER ordinary initatior tRNA for Met is NOT the same as elongation tRNA Met, they differ in sequences.

Shine- Dalgarno sequence

A sequence of purines before the initatior AUG on prokaryotic mRNA. Is a ribosome binding site, to align the ribosome at the initator AUG

Cis acting element for translation initiation in Euk

The 40S ribsome directly binds to the 5’cap and scans for AUG within the Kozak sequence. The Kozak sequence is the sequene located in the 5’ cap that has AUG located innit

43S ribosome complex

The small subunit of ribosome plus the initiator tRNA. 43S complex scans mRNA to find initator codon AUG in the Kozak sequence.

Translation termination

Prokaryotes:

RF1: UAA, UAG

RF2: UAA, UGA

RF3: GTPase that helps RF1 and RF2 form

Eukaryotes:
eRF1: recognizes all UAA, UAG, UGA

eRF3: GTPase that helps eRF1

Cute little note for u sof: There is no eRF2 in euk because the eRF3 is trying to match the function of prokaryotes so that you do not get confused, how nice.

Hai

your doing Great

Polyribosome

A group of ribosomes that translate the same mRNA at a time

Translation initiation in Eukaryotes 4 steps. RSIL

1. Ribosome recruitment: The small subunit of the ribosome 40S binds to the 5’cap with the help of eIF4F

2. Scanning for the start codon: The ribosome scans along the kozak sequence at the 5’cap to find AUG.

3. Initiator tRNA binding: The initiator tRNA Met-tRNA binds to the AUG start codon help of eIF2-GTP

4. Large subunit assembly: The 60S subunit joins forming the 80S initation complex, initation factors are released.

Where does translation of mRNA take place?

In the cytosol or on the endoplasmic reticulum

• cute little note: ribosomes binding on the ER make it appear rough.

Examples of protein

1. Collagen and keratin are structural proteins, assosiated with skin, connective tissue, and hair of organisms

2. Actin and Myosin: are contractile proteins found in abundance in muscle tissue

3. Tubulin: the basis of the function of microtubules in mitotic and meiotic spindle fibers. (Pull chromosomes apart during cell division)

4. immunoglobulins: immune system of vertebrates

5. Transport proteins: Movement of molecules across membranes

6. Hormones and their receptors: Regulate chemical activity

7. Histones: bind to DNA

8. Transcription factors: regulate gene expression

PTM (post translational modifications)

Modifications of amino acids of any proteins by adding extra functional groups, peptides, carbohydrates, lipids or irreversible cleavage of proteins.

Significance of PTM

Protein stability, activity, localization, and molecular interaction.

• Unwarrented however can lead to diseases

Reversible PTM

1. addition of biomolecules

2. addition of chemical functional groups
   
   Rev. BCC

Irreversible PTM

1. cleaveage activation of proteins

2. amino acid modification
   
   Irreversible CACA

Endosymbiosis

Early euk cell engulfed aerobic bacteria, slowly that bacteria evolved as mitochondia inside the Eukaryotic cel.

Mitocondrian genetics

Circular, No histones, many genome copies, No introns

Heteroplasmy

Within the same mitocondria, one or more copies of mtDNA may or may not be mutated.

Mitochondrial DNA encodes

37 genes, 13 proteins for oxidative phosphorylation, 22 tRNAs, 2rRNAs.

Mitochondrial DNA replication peices

• Nuclear DNA polymerases do not replicate mtDNA

• mtDNA POLY: Mitochondrial DNA polymerase

• TWINKLE: Helicase

• mtSSB: single stranded DNA binding protein

mtDNA replication

1. replication begins at OH (heavy strand replication)

2. New L strand synthesis displaces parental H strand

3. Replication exposes OL (Origin of light strand replication

4. H strand synthesis initiates in opposite direction

5. replication proceeds un til complete circular genomes form

Transcription in mtDNA

-POLRMT: mitochondrial RNA polymerase

• TFAM: Mitochondrial transcription factor A

• TFB2M: Transcription initiation factor

• TEFM: Promotes transcription elongation

Human mtDNA has two major promoters: LSP and HSP

• Mitochondria: Promoter → enormous polycistronic transcript → RNA processing → Individuals RNA

does mitochondrial DNA undergo splicing

no

mtRNA processing

the tRNAs act as punctuation points where enzymes come in and cut around it.

• PNaseP cuts at the 5’ end

• ELAC2/ RNaseZ cuts at the 3’ end.

After cleavage many mitochondrial mRNAs recieve

3’ poly A tails.

Mitochondrial translation

Translation occurs in the mitochondrial matrix.

• Mammalian mitroribosomes 55S= 28S small +39S large

Charge of protein and what it means

Positive: Basic

Negative: Acidic

Zwitterionic (neutral)

Sequence Independent Detection assay

• This type does not search for one speciifc protein. This method detects any and all proteins in a sample

• The UV absorbance is 280nm. Proteins naturally absorb this light.

• After seperating proteins by size through gelelectrophorisis scientists add dye, blue and silver so it pops out.

• Mass spectrometry is next, it measures the Mass/Charge ratio, and sequence determination.

UDM (U DA MANNN)

Sequence dependent Detection assay.

1. Western blot and ELISA: antigen recognition via antibody+enzyme reaction

2. Immunofluorescence/ flow cytometry/imaginingL Fluorescent molecule-tagged protein or antibody recognizes specific antigen.

• This method is used to hunt down one specific protein in a crowd.

UV nm and what it is for

Protein: 280 nm

Nucleic acids: 260 nm

Sugar salts and organic solvents: 230nm

Bradford assay

The bradford assay measures protein concentration by detecting a color change in a dye that binds to proteins in solution. The color of the dye is red.

• When the dye binds to a basic amino acid or aromatic residues in protein, it shifts from a red (cationic form to the the blue (anionic form)

• Aidan likes blue anionic.

Bradford measures protein concentraton by color change

Bicinchoninic acid assay

1. Protein and Cu2+ form to give product of Cu+. Reduction of Cu2+ to Cu+

2. Formation of BCA/Cu1+ complex. weird slim looking thing.

The darker the color of purple the greater amont of proteins.

Applications of gene editing

1. medical application: CRISPR: in treating genetic diseases. CAR-Tcells therapy: gene editing of T cells to expiress CAR, to help immune Cells target and destroy cancer cells.

2. Agricultural Applications: GMO for crops, pest resiliant plants improving food quality.

3. Gene drives: Controlling vector borne diseases like malaria through mosquito population modification.

COnsequences of gene editing

• Substitution mutation: Change in onenucleotide

1. Silent mutation: No change in the amino acid: WILL NOT Change function

2. Missense Mutation: Changes one amino acid in the protein. WIll change the function

3. Nonsense Mutation: Converts a codon into a STOP.

Point mutation: Change in one nucletoide

• Interstions and deletions

1. Insertion: addition of one or more nucleotides

2. Deletion: removal of one or more nucleotides

3. Frameshift: Insertions or deletions not in multiples of three, disrupt the reading frame.

Most utalized gene editing techniques in current era CBP

come bring peni

1. CRISPR - Cas9: Single guide RNA +WT Ca9 endonuclease

2. Base editing: Single guide RNA + Dead Cas9

3. Prime editing: Special single guide RNA + Patial dead Cas9

CRISPR in clusters

The sequeces appear as groups of clusters of repeated DNA requences within the bacterial genome

CRISPR Regularly interspaced

The clusters repeats are spaced at regular intervals with unique spacer sequences

• The spacers are phage or plamid DNA fragments from past infections, they act for bacterial defense against future infection

CRISPR SPR Short palindromic repeats

The repreats are short (20-40 bp) and are partially palindromic, meaning they can form hairpin strucutes in RNA.