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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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By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

The Genetic Code

  • Cracking the code

  • Mid 1960s, defined codons

  • Repeating copolymers


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

The Genetic Code

  • Nearly universal

  • Very well conserved but some subcellular organelles show variation


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • Anatomy of an mRNA

  • Eukaryotes

    • Ribosomes recruited via 5’ cap

    • Scanning

    • Poly-A tail enhances translation


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • tRNAs

  • Modified bases created post-transcriptionally

  • Pseudouridine (ΨU)

  • Dihydrouridine (D)

  • Hypoxanthine, inosine, methylguanosine, thymine


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • tRNAs

  • ΨU-loop

  • D-loop

  • Acceptor arm

  • Anticodon loop

  • Variable loop – 3-21 nt

  • 3D structure is L-shaped


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • tRNA charging

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

  • Aminoacyl-tRNAsynthetase


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • tRNA charging

  • Synthetases must

    • Recognize correct tRNA

    • Recognize correct amino acid

  • Amino acid recognition

    • Coarse recognition via size/chemical groups


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • Ribosomes

  • RNA components also measured using S


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • Ribosomes

  • The ribosome cycle


Translation1

Translation

  • Polyribosomes

Prokaryote

Eukaryote

Note the difference –

Due to presence/absence of

nuclear membrane


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • Ribosomes

  • Codon-anticodon interactions in the small subunit

  • Structural tutorial part III


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • Ribosomes

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • Translation initiation

  • Three events:

    • Ribosome recruitment

    • Start codon positioning

    • tRNA brought to P site

  • Different processes in prokaryotes and eukaryotes


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

Translation

  • Translation initiation

  • Prokaryotes

  • Base pairing positions the small subunit


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

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


  • By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    Translation

    • Translation initiation

    • Eukaryotes

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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    Translation

    • Translation elongation

    • Prokaryotes

    • Three steps:

      • tRNA binding at A site

      • Peptide bond formation

      • translocation


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    Translation

    • Translation elongation

    • Eukaryotes

    • Three steps:

      • tRNA binding at A site

      • Peptide bond formation

      • translocation

    • Analogous processes and players


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    Translation

    • Translation termination

    • Release factors (RF)

    • Class II – stimulate dissociation of class I factors

      • RF3, eRF3

      • GTP binding and hydrolysis


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    Translation

    • Translation termination

    • Ribosome recycling

    • RRF – ribosome recycling factor

      • Recruits EF-G-GTP

      • Ratchets ribosome apart

    • All illustrated via animation on website


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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)


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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


    By the end of this series of slides you should be able to explain much of this animation crocoduck bch msstate

    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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