Translation

Background information

Codon Generalities

Degeneracy - is the third position changes, it will still code for the same amino acid, helps prevent major malfunctions

Structure of DNA was proposed in 1953 thanks to Rosalind Franklin's work

tRNA's are charged with AA's

4 nt's make 20 aa's with 3 nt codons

AUG = Start codon = methionine

most amino acids have 2+ codons

tryptophan and methionine only have one codon

3 stop codons

TAG

TAA

TGA

ORF = open reading frame, protein coding sequence, NO STOPS

not all codons are used with the same frequencies across species

proteins = AA + AA + AA + ...

AA's are bound via peptide bonds

tRNA's (aka adapters) bind to RNA and carry amino acids to the ribosomes

Ribosomes = RNA + proteins

catalyze peptide bond formation

code is almost universal

Table is provided on the test

How to read codons

5' to 3' direction

start at start site

Start site = where ribosome attaches to mRNA and incorporates amino acids

3 nt = 1 aa

Bacterial start site is approximately 10nt downstream of ribosome binding site

Eukaryotic start site is at the 5' cap

ribosome scans before translating

if there are lots of stops or the ORF is really short, you need to shift your reading frames

Mutations

missense - changing of codon codes for another amino acid, NOT A STOP

silent - codon changes but amino acid stays the same

nonsense - changing a coding codon to a stop codon

frameshift - adding/deleting 1-2 nt's to change the reading frame and cause premature stops or code for new proteins

Wobble - if third position changes in a codon, it is still recognized by a tRNA that codes for the same amino acid, aka there is flexibility with base-pairing

tRNA's

Most organisms only have 45 of them but some recognize multiple codons

image

Inosine (I) is a base that can pair with U, C, or A which allows one tRNA to pair with three codons

image

MEMORIZE tRNA CLOVERLEAF STRUCTURE

tRNA structure

74-95nt

Parts

discriminator region/acceptor arm

3' end

OH

amino acid arm

sequence = CAA

pseudouridine loop

variable loop - the size is what varies

Anticodon loop

D-loop

contains the anticodon (read from 5' to 3' just like codons so don't forget to flip the order on the test)

Ex: Codon is AUG. Anticodon is therefore CAU.

contains the anticodon

mRNA has codon sequence

no such thing as a stop tRNA

Aminoacyl tRNA Synthetase

20 types of aminoacyl tRNA synthestases

one for each amino acid

1 aminoacyl tRNA synthetase recognizes all the tRNA's for one amino acid

Catalyzes two reactions

adenylation

tRNA charging

accounts for 15% of all RNA

has strong promoters

amino acid + ATP -----> aminoacyl AMP

diphosphate leaves

Class I Reaction

Class II Reaction

Enzyme = class I aminoacyl tRNA synthetase (aka: phe-tRNA synthetase)

2' OH of uncharged tRNA attacks adenylated AA (aminoacyl AMP) at the carbonyl group and releases AMP

Enzyme = class II aminoacyl tRNA synthetase (aka: Gln-tRNA synthetase)

3' OH of uncharged tRNA attacks adenylated amino acid (aminoacyl AMP) at the carbonyl group and releases AMP

Recognition sites of tRNA

discriminator region

anticodon loop (NOT the anticodon itself since it's prone to wobbling)

tRNA nomenclature

Charged example: Met-tRNA^Met

Uncharged example: tRNA^Met

Ribosomes - a type of ribozyme

rRNA makes up 80% of all RNA

ribosomes live in the cytoplasm

slow-growing cells have fewer ribosomes

ribosomes tend to clump together

Ingredients of Translation

Amino Acids (see Degeneracy, and Codon Generalities)

mRNA (see mRNA and RNA Processing Map)

tRNA (see tRNA's)

Ribosomes (see Ribosomes)

Aminoacyl tRNA Synthetases

Accessory Proteins (see Initiation, Elongation, and Termination)

Very energetically expensive (30 ATP's per amino acid), takes 80% of the cell's energy

Translation ALWAYS takes place in the cytoplasm

mRNA

Bacterial mRNA

5' end has a triphosphate

5' UTR has the RBS (Shine-Delgarno sequence), which is the ribosome loading site for 16S rRNA (small subunit)

ORF has AUG start codon

3' end has an OH

Can be polycistronic

Eukaryotic mRNA

5' end has the 7-methyl-G cap and is the ribosome loading site

Has 5' and 3' UTR

Has a 5' and 3' UTR

3' UTR determines mRNA half-life

ORF has AUG start codon

3' end has a poly A tail

ONLY Monocistronic

Subunits

Bacterial

70S = 50S + 30S

Eukaryotic

80S = 60S + 40S

Smaller subunit is the decoding unit

Ribosome = 60% rRNA + 40% protein

proteins tend to 15kDa (small)

Svedberg - the unit of measurement for ribosomal subunits

takes mass and shape into account

ultracentrifugation

charged means to have an amino acid attached

Contact points to tRNA are the acceptor arm and near the anticodon loop

very good at proofreading to avoid mischarging

If so, each ORF has its own RBS (Shine-Delgarno sequence)

Translational coupling - when the stop of one ORF overlaps the start of the next ORF, usually occurs in polycistronic mRNA's, basically ignores the stop codon and continues coding

cap scans mRNA until it finds the start codon (AUG)

rate of synthesis

eukaryotic = 5-10 aa's per sec

bacterial = 20 aa's per sec

3 binding sites for tRNA per ribosome

E site = exit, tRNA is released

P site = peptidyl RNA

A site = incoming aminoacylated tRNA, aka factor binding center, home of elongation and release factors

Elongation

Initiation

Termination

Bacterial

Eukaryotic

1) IF-3 binds to the E site to prevent 50S from binding to 30S.
IF-1 binds to the A site to prevent charged tRNA's from binding. 16S binds to the RBS (Shine-Delgarno sequence)

2) fMet-tRNA^fMet + IF-2 + GTP complex binds to the start codon/P site

3) Large subunit binds to small subunit via hydrolysis reaction. IF factors and IF-2-GTP leave

f = formyl group

formyl group speeds up translation

Simultaneous steps

1) eIF1A blocks the tRNA at the A site. eIF1 prevents the 60S subunit from binding to the E site.

No mRNA yet

2) eIF2-GTP takes the initiator tRNA (Met-tRNAi^Met) to the the P site. eIF5B-GTP and eIF2-GTP stabilize initiator tRNA into the P site

eIF2-GTP +eIF5b-GTP + initiator tRNA = 43S preinitiation complex

Still no mRNA yet

3) eIF4F binds to the 5' cap, brings the mRNA to the preinitiation complex (43S).

RNA helicase (moves the eIF4F) + ATPase scans for the Kozak sequence

Kozak sequence shows ribosome where to settle on mRNA sequence and begin translation

PolyA Tail also binds at this step

eIF4F sticks 5' cap to polyA tail to cause circularization

4) Threading of the mRNA through the 43S subunit occurs by an ATP hydrolysis reaction.

Threading continues until the Met-tRNAi is at the P site.

5) Large 60S subunit can now officially bind to the the 43S preinitiation complex.

All eIF factors are released at this point

Orientation for where we are

The 70S ribosome has the start site (Met-tRNA^Met) aligned with the P site. The next codon is lined up and ready to go for tRNA binding.

1) EF-Tu-GTP (a tRNA chaperone takes a charged tRNA to the A site

2) When chaperoned tRNA with correct BP-ing sequence is brought to the A site, it will bind to mRNA and EF-Tu-GTP is released via hydrolysis

Hydrolysis = GTP -> GDP + Pi

3) Big Scoot!

accommodation - when tRNA in the A site twists to bring the amino acids together to allow peptide bonding

EF-Tu-GTP's can be reused

Peptide bonding occurs in the large 50S subunit

Peptidyltransferase rxn

alpha amino group of A site attaches to carbonyl of P site

mRNA goes to E site

Chain is scooted to P site

Enzyme: EF-G-GTP

imitates EF-Tu-GTP but doesn't have RNA

acts as an A site place holder until big scoot is complete

4) Once scootiong is complete, EF-G-GDP gets kicked out of the A site so the next tRNA can come in

Repeat steps 1-4 until stop codon

Requires a lot of energy

Two Major Parts of Termination

Part One

Part Two

1) RF (release factor) goes into the A site and causes a hydrolysis rxn on the polypeptide chain in the P site

RF's are molecular mimics of tRNA's but the stop codons have a specific binding site for it

2) The polypeptide chain is released from the ribosome. RF3-GDP binds to RF in the A site

3) After GDP is exchanged for GTP on RF3 in the A site and RF leaves

The peptide chain be poking out the ribosome after 7-8 aa's

4) RF3- GTP gets hydrolyzed leading to RF3-GDP product that leaves the ribosome

1) RRF (ribosome recycling factor) binds to the A site

RRF is also a tRNA mimic

2) RRF recruits EF-G-GTP (the translocation factor from elongation + an additional phosphate)

3) GTP hydrolysis (GTP-> GDP) moves uncharged tRNA to the E site and the RRF to the P stie. EF-G-GDP leaves the ribosome (and is ready for elongation step 3- hence recycled)

4) RRF being in the P site leads to subunit disassociation. RRF leaves.

5) IF-3 binds to small subunit to prevent re-sticking and initiation begins again.

0) mRNA is already attached to the 16S part of the small subunit before initiation and RBS (shine-delgarno) is already loaded under the P site

RF and RRF can NOT base pair

This puts AUG at start site