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Translation. By the end of this series of slides, you should be able to explain much of this animation http://www.crocoduck.bch.msstate.edu/EMG/Translation_588x392.swf. The Genetic Code. Degeneracy and synonyms Minimizing impacts of mutation Similar codon – silent mutation or

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Translation

By the end of this series of slides, you should be able to explain much of this animationhttp://www.crocoduck.bch.msstate.edu/EMG/Translation_588x392.swf


The Genetic Code

  • Degeneracy and synonyms

  • Minimizing impacts of mutation

    • Similar codon –

      • silent mutation or

      • similar amino acid

Acidic

Basic

Neutral-polar (+Cys)

Neutral-nonpolar (+Pro, Gly)


Translation

  • Codon – Anticodon pairing

  • The first two positions must pair exactly but the third is more relaxed

  • Anticodon U can pair with A or G on mRNA

  • Anticodon I (derived from G) can pair with U, C, or A

  • Allows for fewer required tRNAs

    • Leucine (6 codons) requires only 3 different tRNAs


The Genetic Code

  • Cracking the code

  • Early 1960s

  • Synthetic polynucleotide RNAs + cell extract  amino acid chains

  • Single polynucleotide chains  single amino acid polymers

  • Poly-U – phe

  • Poly-A – lys

  • Poly-G – Gly

  • Poly-C – Pro

  • No method to order specific codons at the time


The Genetic Code

  • Cracking the code

  • Early 1960s

  • Mixed copolymers

  • Vary ratios of two nucleotides to generate mixed polynucleotide chains

  • 2:1 ratio of A:C  lots of CAC, CCA, ACC codons

    •  histidine/threonine prevelance

  • Suggests potential codons for histidine

  • More precision needed


The Genetic Code

  • Cracking the code

  • Mid 1960s, defined codons

  • Nirenberg and Leder (1964) bound specific individual triplets to ribosomes

  • Ribosomes in turn bound individual amino acids

  • Some triplets not efficient binders


The Genetic Code

  • Cracking the code

  • Mid 1960s, defined codons

  • Repeating copolymers


The Genetic Code

  • Three rules

  • 1. Codons are read in a 5’-3’ direction

  • 2. Codons are non-overlapping with no gaps

  • 3. There is a fixed reading frame relative to the start codon


The Genetic Code

  • Point mutations

  • Missense mutations – changing one amino acid to another

  • Nonsense (stop) mutations – change an amino acid to a stop

  • Frameshift mutations – alter the reading frame

  • Suppressor mutations reinstate the correct amino acid chain (at least partially)

    • Back mutations

    • Intragenic mutations – compensatory mutations

    • Intergenic mutations – involves mutant tRNAs


The Genetic Code

  • Nearly universal

  • Very well conserved but some subcellular organelles show variation


Translation

  • Getting the information contained in an mRNA converted into a protein

  • In bacteria, up to 80% of energy devoted to translation

  • Machinery

    • mRNA

    • tRNA

    • Aminoacyl-tRNAsynthetase

    • ribosomes


Translation

  • Anatomy of an mRNA

  • Open Reading Frames, ORFs

  • Bounded by start

    • 5’-AUG-3’ in eukaryotes

  • and stop codons

    • UAG, UGA, UAA

  • Polycistronic vs. monocistronic

  • UTRs

  • Introns and exons


Translation

  • Anatomy of an mRNA

  • Prokaryotes

    • Ribosome binding site (RBS)

    • 3-9 bp upstream of start codon

    • Complementary sequence on 16S rRNA

    • Alterations strengthen or weaken the RBS


Translation

  • Anatomy of an mRNA

  • Eukaryotes

    • Ribosomes recruited via 5’ cap

    • Scanning

    • Poly-A tail enhances translation


Translation

  • tRNAs

  • Adapters b/t mRNA codons and amino acids

  • 75-95 nt long

  • 3’ terminus = 5’-CCA-3’

    • Amino acid attachment

    • Sometimes added post-transcriptionally via CCA adding enzyme

  • Common shared secondary structure


Translation

  • tRNAs

  • Modified bases created post-transcriptionally

  • Pseudouridine (ΨU)

  • Dihydrouridine (D)

  • Hypoxanthine, inosine, methylguanosine, thymine


Translation

  • tRNAs

  • ΨU-loop

  • D-loop

  • Acceptor arm

  • Anticodon loop

  • Variable loop – 3-21 nt

  • 3D structure is L-shaped


Translation

  • tRNA charging

  • Carboxyl group to 2’ or 3’ OH of A from tRNA

  • Aminoacyl-tRNAsynthetase


Translation

  • tRNA charging

  • Synthetases must

    • Recognize correct tRNA

    • Recognize correct amino acid

  • tRNA recognition

    • No common set of rules across all tRNAs

    • One or more discriminator base(s)

    • One or more anticodon base(s)

    • One or more bases in acceptor stem

    • Various other “inside the L” bases

Important recognition sites for some tRNAs


Translation

  • tRNA charging

  • Synthetases must

    • Recognize correct tRNA

    • Recognize correct amino acid

  • Amino acid recognition

    • Coarse recognition via size/chemical groups


Translation

  • tRNA charging

  • Synthetases must

    • Recognize correct tRNA

    • Recognize correct amino acid

  • Amino acid recognition

    • Small aa’s can fit into large pockets and lead to mischarging

    • Editing

    • Second recognition pocket can accommodate small aa’s but not large aa’s

    • Hydrolyze (cut off) aa’s that fit into pocket

    • Process repeated until aa added that can’t fit in pocket


Translation

  • tRNA charging

  • Ribosomes cannot identify mischarged tRNAs

  • 1962 experiment

  • tRNA-ACA normally charged with Cys

  • Develop cell-free extract with Cys-tRNA-ACA

  • Introduce metal catalyst to change charged Cys to Ala

  • Ribosomes cannot recognize mischarged tRNAs

RNA template

UGUGUGUGUG...

polyCys chain

RNA template

UGUGUGUGUG...

polyAla chain


Translation

  • Ribosomes

  • Massive – three+ RNAs and 50 proteins

  • Large and small subunits

    • Large - Peptidyltransferase center

    • Small – decoding center

    • Measured in Svedberg units (S) corresponding to sedimentation velocity

    • Prokaryotic

      • Total - 70S

      • Small – 30S

      • Large – 50S

    • Eukaryotic

      • Total - 80S

      • Small - 40S

      • Large – 60S

  • Part II of structural tutorial


Translation

  • Ribosomes

  • RNA components also measured using S


Translation

  • Ribosomes

  • The ribosome cycle


Translation

  • Polyribosomes

Prokaryote

Eukaryote

Note the difference –

Due to presence/absence of

nuclear membrane


Translation

  • Ribosomes

  • Three tRNA binding sites

  • A-site – aminoacyl-tRNA

  • P-site –peptidyl-tRNA

  • E-site – exit

  • 3’ termini of A and P tRNAs very close

  • Structural tutorial part III


Translation

  • Ribosomes

  • Codon-anticodon interactions in the small subunit

  • Structural tutorial part III


Translation

  • Ribosomes

  • Channels through ribosome allow mRNA entry/exit, tRNA entry/exit, and polypeptide exit


Translation

  • Translation initiation

  • Three events:

    • Ribosome recruitment

    • Start codon positioning

    • tRNA brought to P site

  • Different processes in prokaryotes and eukaryotes


Translation

  • Translation initiation

  • Prokaryotes

  • Base pairing positions the small subunit


Translation

  • Translation initiation

  • Prokaryotes

  • Initiation factors

  • IF1 – blocks tRNAs from binding to A-site

  • IF2 – escorts initiator tRNA, GTPase

  • IF3 - blocks large subunit from small subunit


Translation

  • Translation initiation

  • Eukaryotes

  • Four steps:

    • Binding of initiator tRNA

    • Auxiliary factors bind to mRNA

    • Bound ribosome scans for start codon (1st AUG)

    • Large subunit recruited

  • 1. eIF1, eIF1A, eIF3, & eIF5 associate with small subunit

    • Analogous to IF1 & IF3

  • 2. eIF2 escorts initiator tRNA


  • Translation

    • Translation initiation

    • Eukaryotes

    • Four steps:

      • Binding of initiator tRNA

      • Auxiliary factors bind to mRNA

        • Cap recognized by eIF4E

        • eIF4A, eIF4G

          • Helicase activity of eIF4A activated by eIF4B

      • Bound ribosome scans for start codon (1st AUG)

      • Large subunit recruited


    Translation

    • Translation initiation

    • Eukaryotes

    • Four steps:

      • Binding of initiator tRNA

      • Auxiliary factors bind to mRNA

      • Bound ribosome scans for start codon (1st AUG)

      • Large subunit recruited


    Translation

    • Translation initiation

    • Eukaryotes

    • Four steps:

      • Binding of initiator tRNA

      • Auxiliary factors bind to mRNA

      • Bound ribosome scans for start codon (1st AUG)

      • Large subunit recruited

    • 1. ATP dependent movement of small subunit toward start codon

    • 2. Codon recognized by anticodon

    • 3. Release of eIF1, eIF2, eIF4B, eIF5 mediated by hydrolysis of eIF2-GTP

    • 4. eIF5B-GTP recruited by eIF1A


    Translation

    • Translation initiation

    • Eukaryotes

    • Four steps:

      • Binding of initiator tRNA

      • Auxiliary factors bind to mRNA

      • Bound ribosome scans for start codon (1st AUG)

      • Large subunit recruited

    • 1. eIF5B recruits large subunit

    • 2. GTP hydrolysis releases eIF1A and eIF5B


    Translation

    • Translation initiation

    • Eukaryotes

    • mRNA is circularlized via a protein bridge b/t eIF4G and PABP


    Translation

    • Translation elongation

    • Prokaryotes

    • Three steps:

      • tRNA binding at A site

      • Peptide bond formation

      • translocation


    Translation

    • Translation elongation

    • Prokaryotes

    • Three steps:

      • tRNA binding at A site

      • Peptide bond formation

      • translocation

    • EF-Tu – escorts tRNAs to A-site

    • GTP hydrolysis reduces affinity of EF-Tu for tRNA

    • Hydrolysis via GTPase domains on ribosome and EF-Tu


    Translation

    • Translation elongation

    • Prokaryotes

    • Three steps:

      • tRNA binding at A site

      • Peptide bond formation

      • translocation

    • EF-Tu – escorts tRNAs to A-site

    • GTP hydrolysis reduces affinity of EF-Tu for tRNA

    • Hydrolysis via GTPase domains on ribosome and EF-Tu

    • Incorrect codon/anticodon match does not position GTPase domain correctly


    Translation

    • Translation elongation

    • Prokaryotes

    • Three steps:

      • tRNA binding at A site

      • Peptide bond formation

      • translocation

    • Accommodation – rotation of the acceptor end of tRNA to bring aa into proximity with peptide chain

    • Some amino acid participation in peptide bond formation but primarily RNA driven


    Translation

    • Ribosomes

    • The peptidyltransferase reaction

    • During aa chain growth, two charged amino acids are housed in the ribosome

      • Peptidyl-tRNA – carries the aa just added to the chain; still attached to the tRNA

      • Aminoacyl-tRNA – the next one to be added

    • Peptidyltransferase reaction breaks tRNA-aa bond on peptidyl-tRNA

    • RNA is the catalyst


    Translation

    • Translation elongation

    • Prokaryotes

    • Three steps:

      • tRNA binding at A site

      • Peptide bond formation

      • translocation

    • Translocation driven by EF-G, peptidyltransferase reaction, and GTP hydrolysis

    • Peptidyltransferase shifts acceptor end into P site but not anticodon end

    • EF-G enters empty factor binding site

    • Hydrolysis changes EF-G conformation to push tRNA out of A site

    EF-Tu + tRNA

    EF-G


    Translation

    • Translation elongation

    • Eukaryotes

    • Three steps:

      • tRNA binding at A site

      • Peptide bond formation

      • translocation

    • Analogous processes and players


    Translation

    • Translation termination

    • Release factors (RF)

    • Class I – recognize stop codons and trigger release of polypeptide

      • RF1 – UAG, UAA; RF2 – UGA, UAA

      • eRF1

  • Peptide anticodon

  • GGQ (gly,gly,glu) motif likely extends into peptidyltransferase center

  • tRNA, RF1


    Translation

    • Translation termination

    • Release factors (RF)

    • Class II – stimulate dissociation of class I factors

      • RF3, eRF3

      • GTP binding and hydrolysis


    Translation

    • Translation termination

    • Ribosome recycling

    • RRF – ribosome recycling factor

      • Recruits EF-G-GTP

      • Ratchets ribosome apart

    • All illustrated via animation on website


    Translation

    • Translation difficulties

    • Nonsense mediated decay

    • Nonsense codon = early stop codon

    • Normal mRNA

      • exon junction complexes displaced by ribosome

    • Nonsense mRNA

      • Exon junction proteins not displaced

      • Recruit Upf proteins to remove 5’ cap

      • Endonuclease degrades mRNA


    Translation

    • Translation difficulties

    • Broken mRNAs lead to stalled ribosomes

    • Stop codon necessary for ribosome release

    • tmRNA – part tRNA, part mRNA

    • ssrA RNA – 457 nt

      • Includes tRNA-like region followed by 10 codons and stop codon

    • Resulting ‘tagged’ protein is degraded


    Translation

    • Translation difficulties

    • Nonstop mediated decay

    • No stop codon results in translation through poly-A tail

    • Poly-lysine peptide and stalling of ribosome

    • Ski7 dissociates ribosome and recruits exonuclease

    • Polylysine protein degraded


    Translation

    • Translation-level control of gene expression

    • 3 aspects of translational-level control

      • A. Localization of mRNAs to certain sites within a cell

      • B. Controlling whether or not an mRNA is translated and, if so, how often

      • C. Controlling the half-life of the mRNA, a property that determines how long the message is translated

    • Mechanisms usually work via interactions between mRNAs & cytoplasmic proteins


    Translation

    • Translation-level control…

    • Cytoplasmic localization of mRNAs

      • Example: the fruit fly, anterior-posterior axis

        • 1. Axis formation is influenced by localization of specific mRNAs along same axis in the oocyte

        • 2. Bicoid mRNAs preferentially localized at anterior end; oskar mRNAs preferentially localized at opposite end

        • 3. Protein encoded by bicoid mRNA is critical for head & thorax development; oskar protein is required for formation of germ cells, which develop at posterior end of larva

        • 4. Localizing mRNAs is more efficient than localizing their corresponding proteins, since each mRNA can be translated into large numbers of protein molecules


    Translation

    • Translation-level control…

    • Controlling mRNA translation

    • Example: mRNAs stored in unfertilized egg are templates for proteins synthesized during the early stages of development;

      • rendered inactive by association with inhibitory proteins

      • Activation of these stored mRNAs involves at least two distinct events:

        • 1. Release of bound inhibitory proteins

        • 2. Increase in length of poly(A) tails by action of an enzyme residing in egg cytoplasm


    Translation

    • Translation-level control…

    • Controlling mRNA stability

    • The longer an mRNA is present in cell, the more times it can serve as template for polypeptide synthesis

      • c-fos mRNA made in response to changes in external conditions in many cells; degraded rapidly in cell (half-life of 10 - 30 min); involved in cell division control

      • In contrast, dominant cell protein mRNAs in a particular cell, like those for hemoglobin, (half-life >24 hours)


    Translation

    • Translation-level control…

    • Controlling mRNA stability

    • mRNA longevity is related to length of poly(A) tail

      • 1. Early study - mRNAs lacking poly(A) tails are rapidly degraded after injection into cell, whereas same mRNA with poly(A) tail is relatively stable

      • 2. Typical mRNA has ~200 adenosine residues when it leaves nucleus

      • 3. Gradually reduced in length as it is nibbled away by poly(A) ribonuclease

      • 4. No effect until the tail is reduced to ~30 A residues; once shortened to this length, the mRNA is usually degraded rapidly


    Translation

    • Translation-level control…

    • Controlling mRNA stability

    • Tail length not the whole story;

      • mRNAs starting with same size tail have very different half-lives –

      • 3' UTR plays role

      • 3'-UTR of α-globin mRNA contains a number of CCUCC repeats that serve as binding sites for specific proteins that stabilize mRNA; if these sequences are mutated, the mRNA is destabilized

      • Short-lived mRNAs often contain destabilizing sequences (AU-rich elements; AUUUA repeats) in their 3' UTR; thought to bind proteins that destabilize mRNA


    Translation

    • Post-translation control…

    • Controlling protein stability

    • Every protein is thought to have characteristic longevity (half-life) or the period of time during which it has a 50% likelihood of being destroyed

      • A. Some enzymes (those of glycolysis or erythrocyte globin molecules) are present for days to weeks

      • B. Other proteins required for a specific, fleeting activity (regulatory proteins that initiate DNA replication or trigger cell division) may survive only a few minutes

      • C. All of the proteins, regardless of expected survival time, are degraded by proteasomes

      • D. Factors controlling a protein's lifetime are not well understood


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