Translation

Replication, Transcription, Translation Synthesis and Template Directions

Replication: DNA synthesized 5’ → 3’ off a DNA template that is read 3’ → 5

Transcription: RNA is synthesized 5’ → 3’ off a DNA template that is read 3’ → 5’

Translation: Proteins are synthesized N → C off an mRNA template that is read 5’ → 3’

Features of the Genetic Code

unambiguous (only one amino acid per codon), degenerate (not only one codon per amino acid), wobble base pairing, only 61/64 codons specify amino acids (3 termination, one initiation)

Unambiguous and Degenerate Genetic Code

only one amino acid per codon but more than one codon per amino acid

Synonymous Codons

multiple codons for many amino acids

Wobble Base Pairing

first two nucleotides of a codon are often enough to specify a given amino acid (one anticodon can recognize more than one codon depending on what the last position is, which saves energy)

Codons Specification

61/64 specify amino acids, 3 specify termination (STOP codon), 1 specify initiation (START codon)

Point Mutations

Silent mutation, missense mutation, nonsense mutation

Silent Mutation

encodes for the same AA

Missense Mutation

encodes for different AA (may not always have sim properties)

Nonsense Mutation

changing to a stop codon (ends the protein early)

tRNA function

translator that reads the mRNA transcript and helps create protein from it

tRNA structure

80 nucleotides long and drawn as clover leaf, but is L shaped in reality and has lots of modified bases, two different parts (acceptor stem/AA arm, anticodon arm)

tRNA Acceptor Stem/AA arm

AA covalently attaches to the tRNA at the 3’ end of this stem

Anticodon Arm

contains anticodon (three base sequence binding to the complementary codon in mRNA)

nomenclature for tRNA

tRNA is named for the amino acid that they typically carry (ex: tRNA^Phe)

tRNA Anticodon/Codon Base Pairing Rules

mostly governed by W/C rules (A-C, G-C) but there are a few wobble pairs that allow one amino acid to be matched to different codons at the 3’ codon (5’ anticodon) position (flexibility in base pairing, wobble position)

Wobble Pairs

G: U

I: A, C, or U

Inosine

an additional nucleotide that can form wobble base pairs with A, C, or U

Inosine

Reasoning behind Wobble Pairs

the 3’ position is less important and there are a few different codons that correspond with the same amino acid that differ only by the third position, so, correspondingly, to save energy, the anticodon has flexible base pairing to recognize all of those codons as opposed to following strict, W/C pairing rules

separate/combined AA and tRNA^AA names

uncharged tRNA and unactivated AA versus charged tRNA and activated AA

Aminoacyl tRNA Synthetases

uses ATP to combine the AA and its corresponding tRNA by covalently attaching AA to the 3’ end of each tRNA (via ester bond at 3’ OH) to make aminoacyl tRNA (AA-tRNA^AA) (at least 20 different ones in different organisms) (uses 2 ATP)

tRNA activation steps

Amino Acid (AA) is stable and floating around so ATP comes around and the AA attacks the ATP causing it to become AA-AMP + 2P_i (which is where the 2 ATP comes from) and that is reacitve, so the tRNA attacks the AA removing the Amp and maing the aminoacyl tRNA bond and having AMP at the end

Kinetic Fidelity

second step is slow because them it gives the tRNA time to change its mind if its been incorrectly paired

Ribosome (Prokaryote vs Eukaryote)

is a ribozyme (made up almost entirely of RNA)

Bacteria: 70S = 50S + 30S

Eukaryotic: 80S = 40S + 60S

Ribosome Components

has three binding sites for tRNA: A-site, P-Site, E-site

Bacterial Translation Factors

Initiation: IF1, IF2, IF3

Elongation: EF-Tu, EF-Ts, EF-G, RF3

IF1, IF3

binds IF1 and IF3 to the 30S subunit, and then the mRNA to initiate

IF2

binds to the 30S subunit and recruits fMet-tRNA^fmet to base pair with teh start codon to start initiation (stops 70S from binding too)

EF-Tu

carries next AA-tRNA into the A-site while also protecting AA-tRNA covalent connection, making sure uncharged tRNA is not in A site and making sure correct AA-tRNA is in the A site

EF-Ts

participates in recharging EF-Tu with GTP

EF-G

facilitates translocation of the AA-tRNA^AA

RF

release factor, binds to A site when STOP codon is present and then causes everything to dissociate

Initiation Site

Shine-Dalgarno sequence of the mRNA binds to the rRNA of the 30S and positions the start codon in the P-site

Bacterial Initiation

1) the 30S subunit binds to the IF1 and IF3 and then mRNA

2) Shine-Dalgarno sequence aligns with the 30S so that the start codon is at the P-site

3) IF2-GTP binds to the 30S subunit and recruits fMet-tRNA^fmet and then helps it move into the P site and base pairs with start codon

4) 50S subunit associates, IF2 hydrolyzes GTP and IF1, IF2, and IF3 dissociate, leaving the 70S initiation complex (ready for elongation!)

Eukaryotic Initiation

lots more initiation factors, 5’cap and poly(A) tail are what orient the ribosome for synthesis (no recognition sequence, just starts at the first AUG after 5’ cap) and no formyl methionine, regular methionine starts protein

Bacterial Elongation

1) EF-Tu brings in the next AA-tRNA to the A site (while also protecting AA-tRNA covalent connection, making sure uncharged tRNA is not in A site and making sure correct AA-tRNA is in the A site) (hydrolyzes GTP in this process)

2) EF-Ts makes sure that EF-Tu is replenished by helping it bind with GTP

3) now the next amino acid is in the A-site and the old one is in the P-site. the new amino acid attacks the amino acid in the P site, creating a peptide bond to make a growing peptide chain, and the old amino acid is transferred onto the new amino acid (catalyzed by ribozyme) (no energy used because energy comes from changing ester to amide)

4) EF-G sits into the A site and pushes everything over (hydrolyzes GTP in this process) and leaves

5) process repeats

Eukaryotic Elongation

same process, just different cofactors (called eEFs)

Bacterial Termination

1) RF1 or RF2 recognizes that there’s a stop codon in the A site

2) water attacks the protein chain in the P site and releases it from the tRNA

3) ribosome dissociates E

Eukaryotic Termination

just one release factor in eukaryotic termination

Affinity of P site vs A site

affinity of ribosome for the P site is higher than that of the A site (because P site is the growing chain, want to keep it and also want to allow the wrong AA tRNA to leave)

Energetics of Protein Synthesis

AA Activation: 2 ATP

Initiation: 1 ATP

Elongation: 2 ATP

so for a protein: (# of AA x 4) - 1 (399 for a 100 AA protein)

Prokaryotic Gene Expression, Why Its Fast

no compartmentalization or mRNA processing; protein synthesized while mRNA still being synthesized; more than one RNA polymerase on a gene; more than one ribosome on mRNA

Eukaryotic Gene Expression

nucleus and mRNA processing (made/processed in nucleus, teranslated in cytosol) but more than one RNA polymerase on a gene and more than one ribosome on the mRNA at a time (like the prokaryote)

Protein Modification

Either Co-Translation or Post-Translation

Post Translational Modifications

deformylation of N-terminal methionine (prokaryotes only)

removal of the N-terminal methionine/formal-methionine

phosphorylation/acetylation (for regulation)

formation of disulfide bonds

addition of carbohydrates (glycoproteins)

Antibiotics and Toxins

antibiotics target bacterial ribosomes, toxins target eukaryotic ribosomes

Frameshift Mutations

Insertion, Deletion

Insertioni

Insertion/Deletion

inserting/deleting a new AA that shifts over the codons after it and disrupts the entire protein

Diseases Caused by Missense Mutations

Alzhemiers, Cystic Fibrosis, Sickle Cell, Thalassemia, Cancer

Altered Hb to Combat Malaria

weakens red blood cells so they cannot support the malarial parasite

Sickle Cell

Hb S, Glu6 to Val Mutation (E6V) in B subunit that locks in the T state

Thalassemia

too few A or B subunits (less Hb A, a2B2), mutations causing an issue with gene expression (different mutations across the globe)

Locations of Different Mutations

Point Mutations: promoter, before/after 5’ UTR/3’ UTR (not too bad), intron (may cause splicing issues), start codon, nonsense mutation (very bad!)

Frameshift Mutations: changes reading frame (very bad)

B-Thalassemia Types

B⁰, B⁺, B⁺⁺ (absence of B subunit, a reduced amt, a normal amt)