Biochem lecture 22: translation

Characteristics of the genetic code

a nonoverlapping triplet code

Nonoverlapping code

Each three-nucleotide triplet is distinct.

Overlapping code (ex. reading ABC, BCD, CDE) (a single base can…)

a single base can be part of multiple consecutive codons

nonoverlapping code

overlapping code

Codes examples with possible # of AAs for each

single base -> 4 (4¹), two bases -> 16 (4²), three bases -> 64 (4³)

Possible # of AA’s

4, 16, 64

For 20 amino acids at least __ nucleotides are necessary per codon

3

____ reading frames are possible because the code is nonoverlapping

3

Each frame gives a different set of _____

triplets

Selection and insertion result in

frame shifts

Insertion and deletion or three insertions can result in

returning to the reading frame

Deciphering the code-using trinucleotides to bind ____ ____ to ____

charged tRNA to ribosomes

Deciphering the code- 1) using trinucleotides to bind charged tRNAs to ribosomes ex.

UUU-> tRNA (Phe), AAA -> tRNA (lys), CCC -> tRNA (pro)

Deciphering the code- 2) using defined ….

polynucleotides in a cell-free translation system 

How are polyribonucleotides synthesized

chemically or with polynucleotide phosphorylase

Deciphering the code- resulted in the

genetic code table

The genetic code is _____

degenerate

Degenerate

some amino acids have more than 1 codon associated with it

Degeneracy can minimize

the deleterious effects of mutations

3rd position mutations can be ____

silent

____ is the adapter

tRNA

tRNA molecule can do… and this is what type of process

base pairing, selective process

Anticodon and codon are in _____ direction

opposite direction

Codon and anticodon are inherently antiparallel due to what property?

polarity

Watson-Crick base pairs

A-U, G-C

What is a base pair that is also possible that Watson-Crick did not know about

G-U

Wobble hypothesis

the third base of a mRNA codon (at the 3’ end) can pair flexibly with the first base of a tRNA anticodon (at the 5' end), allowing one tRNA to recognize multiple synonymous codons

___ codons possible, ___ codons for stop leaves ___ codons to be recognized only up to __ unique tRNA anticodons discovered

64; 3; 61; 45

we know wobble hypothesis exists because…

there are more codons than anticodons

Wobble pairing allows some ….

tRNA (anticodons) to recognize more than one codon

The wobble hypothesis 1

The 1st two 5' bases of an mRNA codon always form strong base pairs with the tRNA anticodon, conferring most of the specificity.

I pairs with

A, U, or C

The wobble hypothesis 2

The 5’ base of the anticodon determines the number of codons recognized by the tRNA

when the anticodon 5’ base is ___ or ___ only 1 codon is recognized

C or A

When the anticodon 5’ base is ___ or ___ 2 different codons may be recognized

U or G

when the anticodon 5’ base ___ 3 different codons can be recognized

I

The wobble hypothesis 3

When an amino acid is specified by several different codons, the codons that differ in either of the 1st two bases require different tRNAs

The wobble hypothesis 4

minimum of 32 tRNAs (4^2 X 2) are required to translate all 61 codons.

The translation machinery

mRNA, tRNA, ribosomes, soluble protein factors (initiation factors, elongation factors, termination factors)

tRNA is composed of

amino acid arm, Tψ (psi) C arm, Anticodon Arm, Anticodon, D Arm

uridine is found in what part of tRNA

anticodon arm

dihydrouridine is found in what part of tRNA

D arm

pseudouridine (ψ) is found in what part of tRNA?

TψC arm

tRNA 2D structure

cloverleaf

Why do we have tRNA instead of just RNA? (delete this)

because tRNA adds a benefit, regular RNA gets stiff, tRNA is more flexible (Temperature decrease -> motion decrease -> need tRNA)

Difference between uridine vs dihydrouridine

uridine has an aromatic ring (double bond, planar)

Dihydrouridine is more ____ than uridine

flexible

tRNA 3D structure

an L in 3D

Ribosomes are ….

RNA-protein machines

S =

svedberg (sedimentation rate), correlates with size

Eukaryotic ribosome

two subunits, larger than bacterial ribosomes (proteins are larger)

Bacterial ribosome

two subunits, smaller than eukaryotic ribosome

rRNA are key for ________ function

ribosomal

secondary structure of rRNA

folded ribosome structure

Ribosome structure- Ribosomes are made of…

RNA, protein, and a cleft

Assembly of ribosomes in eukaryotes begins in the ____ (location)

nucleus

Assembly of ribosomes in eukaryotes-location goes from where to where

nucleolus -> nucleoplasm -> cytoplasm

Protein synthesis 5 major steps

activation of amino acids, initiation, elongation, termination and ribosome recycling, folding and posttranslational processing

Protein synthesis- stage 1

aminoacylation reaction or tRNA charging

Protein synthesis- stage 1 overall

amino acid + ATP + tRNA <-> aminoacyl-tRNA + AMP + PPi

Protein synthesis- stage 1 Nomenclature

Ser-tRNA^ser charged; tRNA^ser uncharged

Aminoacylation is carried out by

aminoacyl-tRNA synthetase

Aminoacylation-____ here is essential

accuracy

Protein synthesis- stage 2

initiation

Protein synthesis- stage 2 steps

30S binds IF-1 and IF-3, mRNA binds positioned by 16S rRNA, if IF-2 GTP binds 30S subunit, fMet-tRNA^fmet binds, which base-pairs with start codon, 50S binds IF-2 GTP hydrolyzed, IF-1, IF-2, IF-3 released, 70S initiation complex 

An RNA-RNA interaction with ____ ____ positions ____ on the _____ in prokaryotic mRNA

16S rRNA positions the mRNA on the ribosome

The eukaryotic initiation process

scanning to 1st AUG from 5’

The eukaryotic initiation process

factors binding to subunit , propagate along the message until you fit AUG

Protein Synthesis- Stage 3

elongation

Protein Synthesis- Stage 3 steps

1) binding of aminoacyl-tRNA to A site 2) peptide bond formation 3) translocation

Protein Synthesis- Stage 3.1 steps

EF-Tu GTP binds to aa-tRNA^aa, aa-tRNA^aa-EF-Tu GTP binds to A site, GTP is hydrolyzed EF-Tu GDP dissociates, EF-Tu GTP is regenerated

Protein Synthesis- Stage 3.2

peptide bond formation

Protein Synthesis- Stage 3.2 steps

A-site amino acid’s NH2 attacks P-site amino acid -> peptide bond forms -> chain transfers to A site

Protein Synthesis- Stage 3.3

translocation

Protein Synthesis- Stage 3.3 steps

amino acids shift from A binding site to P binding site catalyzed by EF-G-GTP, AUG moves from P to E site

Protein Synthesis- Stage 4

termination

Protein Synthesis- Stage 4 steps

stop codon enters A site -> release factor binds to UAG -> peptidyl-tRNA link hydrolyzed -> peptide leaves -> components dissociate

Protein synthesis is energetically

expensive!

AMP -> AMP + PPi occurs in what step/s

aminoacylation

GTP -> GDP + Pi occurs in what step/s

proofreading and translocation

___ high energy phosphate bonds per peptide bond

4

___ ____ for initiation per protein

1 GTP

Coupling of _____ and ____ in bacteria

transcription and translation

In bacteria, messenger RNA (mRNA) is protected by

ribosomes, acting as polysomes

Bacterial protein synthesis is _________, but eukaryotic protein synthesis is not

co-transcriptional

Protein synthesis-Stage 5

post-translational processing

Stage 5: Post translational processing examples

N- and C- terminal modification, loss of signal sequences, amino acid modification, disulfide bond formation, glycosylation, isolation, addition of prosthetic groups, proteolytic processing

Addition of carbohydrate side chains function and location

plays a key role in protein targeting, occurs in ER

Addition of isoprenyl groups

adds aliphatic chain, anchor otherwise soluble proteins to the membrane

Many antibiotics target

protein synthesis

Ribosomes have three binding sites

A (aminoacyl) site, P (peptidyl) site, and E (exit) site

Antibiotic examples

chloramphenicol, Cycloheximide, Erythromycin, Fusidic acid, Puromycin, Streptomycin, Tetracycline, Diphtheria toxin, and Ricin

Chloramphenicol

Inhibits peptidyl transferase on the prokaryotic large subunit

Cycloheximide

Inhibits peptidyl transferase on the eukaryotic large subunit

Erythromycin

Inhibits translocation by the prokaryotic large subunit

Fusidic acid

inhibits elongation in prokaryotes by binding to EF-G GTP in a way that prevents its dissociation from the large subunit