CH 37: protein synthesis and genetic code

Codon

3 letter code on mRNA

Genetic code

Linear array of codons

Nucleotide sequence of MRNA

61 codons code for 21 Aa

3 nonsense codons

Note: some AA have more than 1 codon that codes for it (Leu, Ser, VAL)

Some have 1 distinct: Met: AUG,Trp: UGG

Nonsense codons

Used as termination signals

UAA UAG UGA

Initating codon

AUG w/c codes for MET

Genetic code feat:

Degenerate

Unambiguous

Non overlapping

Not punctuated

Universal

Degenerate

Multiple codons decode same AA

eg 6 for Serine, 4 for Val

Degeneracy resides on 3rd nucleotide of triplet

Unambiguous

A codon codes for a SPECIFIC AA; no other

Non overlappig

Message is read in a continuous manner with NO PUNCTUATION

- code is commaless

Disad: cant detect if codon inserted can cause mutation

Universal

In nearly all except MT codons; highly conserved

5' to 3'

Direction of reading codons

TRNA: the adapter mol

Atleast 1 trna for each 20 AA

Charged trna

Trna with AA attached to it

Charging

2 step -> recognition and attachment

Aminoacyl trna synthethase

Form an aminoacyl-Amp-enzyme complex

Recognizes a specific TRNA to which it attaches the aminoacyl moiety the 3'HYDROXYL ADENOSYL TERMINAL

Ester linkage

Attachment of AA to specific tRNA

NB

A specific codon, only a specific AA will be incorporated

A Specific AA, more than one codon may bebused

TWC arm

thymidine-pseudouridine-cystidine arm

- involved in BINDING if aminoacyl-trna to ribosomal surface at site of CHON synthesis

D arm

Impt for proper recognition of a given trna species by its proper aminoacyl trna synthethase

Acceptor arm

Loc at 3' OH adenosyl terminal

Site of attachment of SPECIFIC AA

Anticodon arm

Consists of 7 nucleotides, RECOGNIZES 3 letter codon in mrna

Anticodon loop

Sequence is read in 3' to 5' dir.

Consists of a variable base-modified-purine-xyz-pyr-pyr-5'

Antiparallel

Codon of mrna & anticodon of trna

Wobble / wobble position

Degeneracy reside on 3rd letter on nucleotide cause unstable H bonding

-> faster rate of CHON synthesis

Transition & Transversion

Single base changes/ base subs/ point mutations

Effects of single base changes

1 Silent murations - no detecteble defect; most likely at 3rd nucleotide of codon

Wobble: least sensi to change

2 Missense : occur if diff. AA is incorporated at cor. site in CHON mols; mistaken AA

--> acceptable, partially, unacceptable

3 Premature termination with a nonsense codon

- produces omly a fraction of intended CHON mol

Framshift mutation

Deletion/Insertion of 1 nucleotide from the coding strand of a gene-> altered readingg frame in MRNA

Suppressor mutation

Can counteract some mutation effect

Suppressor + trna mol

- Formed as result if alterations in anticodon region; ABN functioning

- Capable of binding to and decoding altered codons

- NOT capable of distinfuishing bet. Normal codon and one from gene mutation

- Results in DEC viability

Protein synthesis

Initation, elongation, termination

Primary structure

Is the one being synthesized; dictated by genetic sequencs; read in 5' to 3'

Initation

A. ribosomal dissociation (40s + 60s)

B. Formation of 43s PIC

C. Formation of 48s IC

D. Role of poly a tail in initation

E. Formation of 80: IC

A. Ribosomal dissociation

- 2 initiation factors, eif-3 & eif-IA bind to newly dissociated 40s subunit

- delays reassociation with 60s subnit

-allow other translation IFs to associate with 40s

B. formation of 43S PIC

see book for steps/notes

eif-2

consists of a,B,y subunits

one of the two control points for protein synthesis

initiation in eukoryotic cells.

Phosphorylates EIF-2

phosphorylated (on serine 51) by at least four

different protein kinases (HCR, PKR, PERK and GCN2) that are

activated when a cell is under stress and when the energy

expenditure required for protein synthesis would be

deleterioius.

Phosphorylated elF- 2α

binds tightly to and inactivates the GTPGDP

recycling protein elF-2β. Thus, preventing formation of the 43s

preinitiation comples and blocking protein synthesis.

Phosphorylates 4E*

Insulin + Mitogenic GF

PKR

SEE NOTES

C. Formation of 48S initiation complex

SEE NOTES

role of poly(a) tail in initation

3' poly (A) tail & its binding CHON, Pab1p: are required for efficient initation of CHON synthesis

- stimulates recruitment of 40s to mRNA

D. formation of 80s subunit

SEE NOTES

4F Complex: 4e+ 4G

Controls rate of PROTEIN TRANSLATION

4F FAMILY!!!

4E (father)

- binds to m7 G CAP of 5' end of mRNA

- resp for RECOGNITION of CAP

- RATE LIMITING STEP

4G (mother)

- Serve as scaffolding CHON that stabilizes complex

- also bind to eif-3(high affinity); also bind to 4a+4b

eif-4A, FB (children)

Binds and reduces complex 2ndary structure (relax CAP) through atp-ase dependent helicase acts

pab1p + eif-4g

binds eif-4E that si bound to cap structure

stabilizes mrna

Eif-3 (fam friend lol)

Links complex to 40s ribosomal unit

binds with high affinity to 4G comp of 4F and links it to 40s subunit. Keeps mRna in place

Insulin + Mitogenic GF

Results in phosphorylation of 4E on Ser209

Phosphorylated 4E

Binds to CAP more avidly -> enhances rate of initiation

CHON that Binds to inactive 4E

4E- BPI (aka PHAS-1)

4E- BP2

4E- BP3

BPI

- Protein that Binds with high affinity to 4E

-effectively inh. Translation initiation

4E•BP1 association

Prevents binding of 4e&4g=4f

Elongation

Cylic process

1 AA is added at a time to nascent peptide chain

Determined by: orders of codon in MRNA

Elongation process

A. Binding of amino-acyl trna to A Site

B. Peptide bond formation

C. Translocation of ribosome to mrna

D. Expulsion of deacylated trna from P to E sites

Energy used for 1 peptide bond

Hydrolysis of:

2 Atp mol to ADP

2 GTP To GDP

Skipped steps

Termination

Stop codon mena: UAA, UAG, UGA (A site)

Releasing factors

CHON that hydrolyze the peptidyl-trna bond

RF-1

Recognizes that a stop codon is in A site

Polyribosome

Aka polysome: multiple ribosome on mrna

No. Of polyribosome on mRNA

correlwtes with length or mRNA

P bodies

- Small dense compartments that incorporate mRNA as mRNP

- site of tanslation repression + mrna decay

MRNP

when mrna are bound by specific packaging CHON + exported out of nucleus

Mechanism for rapid control

Di ko chindi na part

Ferritin

IRon binding CHON that prevents ionized iron from reaching toxic levels

- disruption of protein-mrna interaction -> act. Ferritin mrna -> translation

Encephalomyocarditis

Translated more efficiently

Reovirus/ vesicular stomatitis virus

Replicate efficiently -> abundant

Polivirus, picorna

Selective advantage by disrupting the function of 4F complez

Tetracycline

Prevents binding of aminoacyl trna to A site

Chloramphenicol, macrolides

Bind to 23s

Puromycin

Tyrosynl-trna analog

Inhibits CHON synthesis on ribosomes

Incorporated via A site but causes premature release of polypeptide

Cycloheximide

Inh. Peptidyl transferase in 60s eukaryotes

Presuambly binds to an RNA comp

Diphtheria toxin

Catalyzes adp-ribosylation of EF-2 on diphatamide -> inactivates EF-2

Ricin

Inactivates 28s by providing N-glycolytic cleavage; remocal of a single adenine