6- Genetic codes or translation/ protein metabolism

Protein synthesis

• protein synthesis is highly energy demanding so it is highly coordinated

• can use 90% of the chemical energy of a cell

• number of copies of proteins produced = number of protein needed

• proteins are targeted to cellular locations

• degradation keeps pace with synthesis

molecular coding of protein sequence information - transcription

• in transcription, one strand of double-stranded DNA acts as the molecular template for RNA synthesis (DNA —> Messenger RNA)

• 3 nucleotides code for one “codon”

molecular coding of protein sequence information - translation

• the triplets of nucleotides in mRNA bind to complementary triplets in tRNA

  • the tRNA molecules carry an amino acid assocaited with the particular triplet

  • amino acids are then assembled in peptide chains

What does a protein sequence determine?

biological function

What features of protein synthesis make it a very complex process***

• >70 ribosomal proteins

• ~20 aa activation enzymes

• ~20 protein factors for intiation,elongation, and termination of peptides

• ~100 additional enzymes for final processing

• ~40 kinds of tRNAs and rRNAs

early advances in understanding protein synthesis

1. protein synthesized at ribosomes

2. amino acids activated for synthesis by attachement to tRNA via aminoacyl-tRNA synthetases

3. tRNA acts an adapter to transcribe mRNA into protein (tRNA —> amino acid sequence)

Rough Endoplasmic recticulum

where protein synthesis occurs inside ribosomes

Genetic code for proteins

• consists of triplets of nucleotides

• there are 20 common,.genetically encoded amino acids

• A two letter code in groups of two is insufficient (16) or (4×4)

• A four letter code ingroups of 3 is sufficient (64) (4×4×4)

• living organisms use non overlapping mRNA code with no punctuation

tRNA

• reads the sequence on mRNA 3 nucleotides at a time and serves as an adapter to transcribe mRNA into protein—>amino acids

Is our genetic code over-lapping or non overlapping?

non overlapping —> read 5’—>3’ 3 letter code

Overlapping code

• when one nucletide is shared between the boundaries of the primary mRNA transcripts of two or more genes

What did Nirenberg and Mathaei discover about the genetic code?

• poly(u) and 20 radiolabeled amino acids fed to E.coli —> only *phe produced

• hence, uuu codes for phe

what did khorana discover about the genetic code?

• used to define mRNAs in planned patterns

  • that is (AC)n (alternating ACA and CAC codons —> His and Thr

What amino acids do AAA,UUU,CCC code for?

• AAA= lys tRNA

• UUU= phe tRNA

• CCC= pro tRNA

Features of the Genetic code

• the code is written in the 5’ —> 3’ direction

• 3rd base is less important in binding to tRNA

• The 1st codon establishes the reading frame

  • if reading frame is thrown off by a base or two all subsequent codons are out of order

• 61/64 codons code for amino acids

• there are termination and intiation codons

What are the termination and intiation codons?

Termination:

• UAA, UGA, UAG

Initiation/start codon:

• AUG —> met

Is the genetic code universal?

• yes but there are a few exceptions

• it is used by prokaryotes and eukaryotes across species

wobble

• allows some tRNAs to recognize more that one codon

• the 3rd base of a codon (in mRNA) can form non canonical base pairs with its complement(anti codon) in tRNA

• some tRNAs contain inosinate(I) which can H-bond w/ U,C,and A

  • these H-bonds are weaker and were named by crick as wobble base pairs

• codon written in 5’ - 3’ direction and anti codon is written in 3’-5’ direction

molecular recognition of codons in mRNA by tRNA

• the codon sequence(in mRNA) is complementary w/ the anticodon (in tRNA) sequence

• the codon in mRNA base pairs with the anti codon in tRNA via hydrogen bonding

• the alignment of two RNA segements is antiparallel

• wobble base pairs 1st nucleotide of anticondon to 3rd base pair of codon

What is the ribosomes role in protein synthesis?

• make up 25% of the dry weight of bacteria

• ~65% rRNA, 35% rRrotein

  • rRNA forms the core size: rRNA > rRrotein

  • RNA does the catalysis of peptide bond formation

• Made of two subunits bound together(30s and 50s) in bacteria with/ mRNA running through them

Are the structures of Ribosomes similar in bacteria(Prokaryote) and yeast(Eukaryotic)

• similar sturcture in both pro and euk

• both have rRNA and rRrotein

• in eukaryotes, larger(80s), more complex, contains >80 proteins

• chloroplasts and mitochondria have ribosomes simpler than those in bacteria

Structure of rRNA

• complex secondary structures

• the ssRNA’s have specific 3-D structure w/ extensive intrachain base pairs

• shape of rRNA’s are highly conserved (2-d structure) —> similar between bacteria,archaea, and eukaryotes

Bacterial ribosome

• large + small subunit = 70s

• Large subunit = 50s ( 23S rRNA and 5S rRNA and 36 proteins)

• small subunit= 30s( 16S rRNA and 21 proteins)

Eukaryotic Ribosome

• large + small subunit = 80S

• large subnit = 60S (5S rRNA, 28S rRNA, 5.8S rRNA and 47 proteins)

• small subunit = 40s(18S rRNA and 33 proteins)

Characteristics of tRNA

• ssRNA of 73-93 nucleotides in both bacteria and eukaryotes

• clover leaf structure in 2-D

• “twisted L” shape in 3-D

• must have G at 5’-end, all have CCA at 3’ end

• have modified bases —> methylated bases and so on

• has an amino acid arm, anticodon arm, D arm, TΨC arm

Amino Acid arm( trna characteristics)

• has amino acid esterfied via carboxyl group to the 2’OH or 3’OH of the A of the terminal CCA codon

D arm

• contains dihydrouridine(D)

• contributes to folding

TΨC arm

• contains pseudouridine(Ψ) - has bonding between base and ribose

• helps in folding
  

Overview of the 5 stages of protein synthesis

1. activation of amino acids

   • tRNA aminoacylated(trna carries amino acid)

2. Intiation of translation

   • mRNA and aminoacylated tRNA bind to ribosome

3. Elongation

   • cycles of aminoacyl-tRNA binding and peptide bond formation … until a STOP codon is reached

4. termination and ribosome recyling

   • mRNA and protein dissociate, ribosomes recycled

5. folding and post transitional processing

   • catalyzed by a variety of enzymes

stage 1 of protein sythesis - activation of amino acid

• step 1 creation of aminoacyl intermediate

  • amino-acyl-tRNA synthetases esterify 20 amino acids to corresponding tRNA’s

  • COO- of amino acids attacks phosphate of ATP —> creates aminoacylaldehyde intermediate

  • pyrophosphaste(PPi) is also cleaved so the reaction is driven forward by two phosphoanhydride bond cleavages

  • the fate of aminoacyladenylate varies

• product = aminoacyl tRNA product

Two classes of aminoacyl-tRNA synthetases

• there are 2 classes each in charge of 10 amino acids and final purpose is to add aminoacyl to 3’ end of pentose generating aminoacyl tRNA product

Stage 2: intiation(prokaryotes and eukaryotes)

• the first tRNA is unique

• the first codon of any peptide is AUG(met)

• All organisms have two tRNAs for met

Stage 2 intiation (prokaryotes)

• in bacteria plus chloroplasts and mitochondria intiation of tRNA inserts N-formymethionine(uses a special tRNA fmet)

• interior met is inserted with normal tRNA

• in bacteria initiation requires:

  • 30s ribosomal unit

  • mRNA

  • fmet-tRNA ( is the tRNA carrying first amino acid)

  • initiation factors IF-1, IF-2, and IF-3

  • GTP

  • 50s ribosomal unit

  • mg2+

intiation prokaryotes - step 1

• 30-s Ribosomal subunit binds to IF-1,IF-2, and IF-3 and mRNA

• initiation factor IF-3 keeps 30S and 50S ribosomal subunits apart

• the initiating 5’ AUG codon of mRNA is guided to its correct position by the shine Dalgarno sequence( sequence only in e.coli/prokaryotes) (a region in mRNA that is complementary to a sequence in Ribosomal RNA ) to find AUG

intiation prokaryotes - step 2

• fmet tRNA joins the complex

• Formylmethionine tRNA binds to the peptidyl(p) site along with initiating(5’) AUG

intiation prokaryotes step 3

• 50 S subunit associates

• large 50S subunit combines with the 30S subunit forming the initiation complex

• IF-2 hydrolyzes GTP - end of initiation

Stage 2 -intiation (eukaryotes)

• use more initiation factors

  • over 12, including eIFIA and eIF3(functional homologs of IF-2 and IF-3)

  • Has different mechanistic details

  • has a step that circularizes the mRNA during initiation

intiation eukaryotes - step 3

• mRNA binds with eIF4F

• eIF4E(binds the 5’ CAP)

• eIF4A(an ATPase and RNA helicase)

• eIF4G(linker protein which binds to PABP-poly(A) binding protein at the 3’ poly(A) tail )

stage 2 intiation - step 4 (Eukaryotes)

• scanning of mRNA until an AUG codon is found

stage 2 intiation - step 5 (Eukaryotes)

• 60S subunit assoicates and many of the intiation factors are released

stage 3: elongation(prokaryotic) - step 1

• binding of the incoming second aminoacyl-tRNA

• incoming aminoacyl-tRNA binds first to an ET-Tu-GTP complex

• the aminoacyl-EF-Tu-GTP complex binds to the aminoacyl(A) site of the 70S intiation complex

• after GTP hydrolysis, EF-Tu-GDP

  ~EF-Ts recylcles EF-Tu

stage 3: elongation(prokaryotic) - step 2

• peptide bonds forms

• there are now two amino acids bound to tRNAs positioned for joining

  • one is on the A site, the other on the p site

• N-formethionyl group is transferred from its tRNA in the P site to the amino acid in the A site

  • the reaction is catalyzed by the 23S rRNA(ribozyme)

• “uncharged” (deacetylated) tRNA f met is now in the P site

stage 3: elongation(prokaryotic) - step 3

• translocation of the ribosome

• the ribosome moves one codon toward the 3’ end of the mRNA

  • uses energy from GTP hydrolysis

    • GTP is part of EF-G(translocase)

• leaves A site open for new aminoacyl-tRNA

stage 3: elongation(eukaryotic)

• steps are similar to bacteria

• elongation factors - eEF1α(EF-Tu), EF1βγ(EF-Ts),eEF2(EF-G)

• difference: eukaryotic ribosomes do not have an E site; the uncharged tRNAs are released from the P site

Stage 4: termination

• signaled by a STOP codon

• UAA,UAG, or UGA in the A site will trigger the action of termination factors(release factors) RF-1,RF-2,RF-3

• These help to:

  • hydrolyze terminal peptide-tRNA bond

  • release peptide and tRNA from ribosome

  • cause subunits of ribosome to dissociate so that intiation can begin again

Stage 5 - posttransitional modifcations

• some proteins require modification before the fully active conformation is achieved

• Posttranslational modifications include:

  • enzymatic removal of a formyl group from the first residue or removal of met and sometimes additional residues

  • acetylation of N-terminal residue

• Removal of signal sequences or other regions

• attaching carbohydrates

• removing sequence to activate an enzyme

What stage do antiobiotics and toxins target

translation

Puromyocin

• made by the mold streptomyces alboniger

• similar structure to 3’end of aminoacyl-tRNA

• so it binds to the A site of ribosomes, forming bond with growing peptide

• but can’t participate in translocation and dissociation

• TERMINATES protein synthesis

Tetracylines

• block the A site on the ribosome

chloramphenicol and cycloheximide

• they block peptidyl transfer

• chloramphenicol inhibits mitochondrial and chloroplast ribosomes as well as bacterial

Proteins move from site of synthesis to:

• exit a cell

• become part of the membrane

• enter a subcellular compartment and so on

• most have a signal sequence at or near the N-terminus

  • 13-36 amino acid residues in length

• takes place in eukaryotic cells where subcellular organization aids in compartmentalizing metabolic pathways

Peptides directed to the ER

• as peptide emerges from the ribosome the signal sequence is bound by signal recognition particle(SRP)

• SRP/ribosome/RNA complex is delivered to the ER lumen

  • some modification takes place here(glycosylation…)

• transport vesicles then take proteins to golgi apparatus where protiens are sorted in ways poorly understood

• proteins enter the biosynthetic/secretory pathway - go to where they need to go to complete function

• proteins for mitchondria and chloroplast bind chaperone proteins in the cytosol and are delivered to receptors on the exterior of the organelle

how are proteins targeted for and imported into the nucleus

• proteins for the nucleus have a nuclear localization sequences(NLS)

  • An NLS is not cleaved after the protein is targeted

  • the nuclear envelope can degrade and proteins will need to re-enter the nucleus

• it binds importin α and β and GTPase called Ran

• the complex docks at a pore and is imported

• ribosomal proteins are synthesized in the cytosol, imported back into the nuclease, assembles into subunits in the nucleolus, and the exported back to the cytosol

Protein degradation is inevitable

• half lives of proteins range from seconds to days to even months

  • hemoglobin is long lived - cause we need it for oxygen

  • defective proteins are short - lived as are many metabolism regulatory proteins that respond to rapidly changing needs

  • all are eventually degraded

mechanisms of degradation e.coli

• Lon( for “long form” an atp-dependent protease) hydrolyzes defective or short-term peptides

mechanisms of degradation eukaryotes

• proteins are linked to protein ubiquitin

  • via activating enzyme E1, conjugating enzyme E2, and ligating enzyme E3

• ubiquitinated proteins are cleaved by the 26 proteasome(digest proteins into amino acid so amino acids can be recycled) Complex

• ubiquitin is very highly conserved among all eukaryotes

• ALL PROTEINS ARE EVENTUALLY RECYCLED

genetic code

• 3 nucleotide code for 1 codon