BIO311 Prokaryote Gene Expression Section 4 Structure of ribosomes

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BIO311 - Prokaryote Gene Expression Section
4Structure of ribosomes

• Prof Jasper Rees
• Department of Biotechnology, UWC
• www.biotechnology.uwc.ac.za/teaching/BIO311

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3D view of 50S subunit of Haloarcula marismortui
ribosome
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The Ribosome

• A large RNA and protein particle visualised by EM
  in the cytoplasm of all prokaryotes and
  eukaryotes and in eukaryote organelles
• Frequency associated with mRNA and can also be
  associated with membranes
• Responsible for protein synthesis
• Structure determination now at atomic resolution
  after 40 years of study using many techniques

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Ribosome components

• All ribosomes made up of rRNA and rProteins
• Approximately 66 rRNA and 33 protein
• rRNA is folded into highly conserved secondary
  and tertiary structures
• Proteins bound to RNA core, now found to be on
  the outside of the RNA region
• All have two subunits, with equivalent roles

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Ribosome components
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Study of the ribosome structure - I

• Early studies used negative stained electron
  microscopy
• Image reconstruction required to generate 3D
  models
• Subsequent biochemical and biophysical data
  incorporated with this model
• Immuno-EM allowed identification of specific
  protein positions on the model

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EMs of the ribosome structure

• Early studies used negative stained electron
  microscopy
• Many images used to gain understanding of the
  different views

50S Subunits
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3 dimensional models built from image
reconstruction
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30S and 50S models for E.coli ribosome
30 S subunit
50S Subunit
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30S and 50S together provide a 70S model which
agrees with EM data
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Arrays of ribosomes allow higher resolution image
reconstruction

• Use of 2D arrays (paracrystalline) allows the
  generation of high resolution images since all
  the images are in register as the subunits are
  oriented in the same direction

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Immunoelectron microscopy

• Antibodies raised against specific proteins
  purified from the ribosome
• Specificity of antibodies can be characterised
• Antibodies have two identical binding sites for
  the target protein
• Binding antibodies to isolated ribosome subunits
  will result in formation of dimers
• Assignment of antibody binding sites on the
  surface of the ribosome provides map of surface
  exposure of individual proteins

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Immuno EM data
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Model from Immuno EM data
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Study of the ribosome structure - II

• In vitro reconstitution experiments possible
  because ribosomes self assemble
• Allows the characterisation of interactions and
  binding sites
• Allows incorporation of modified rRNA or protein
• Describes pathway of assembly for both subunits

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Purification of ribosomal proteins

• Purification of ribosomes, is followed by
  disruption and isolation of individual proteins
• Highly basic nature makes purification more
  difficult
• 2D gel electrophoresis provides methods for
  identification of all of the proteins
• Can identify modifications to proteins with 2D
  gels (eg cross-linking reactions)

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Assembly Map for 30 S subunit

• In vitro assembly provides a method of ordering
  interactions between components
• Can map binding sites and binding order and
  dependency
• Have map for 30S and 50S subunits

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In vitro assembly allows labelling of specific
proteins

• Purification of all of the proteins allows the
  individual proteins to be labelled before
  reassembly
• Can label with fluorescent molecules
• Can label whole ribosome with heavy isotopes and
  then incorporate 2 heavy proteins into a
  light ribosome

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Fluorescence Resonance Energy Transfer (FRET)

• Two fluorescent groups with 100Å of each other
  can transfer energy through Resonance Energy
  Transfer mechanisms
• Efficiency of energy transfer inversely
  proportional to D6
• Efficiency can be used to measure molecular
  distances
• If label two proteins in reassembled ribosome
  then can measure distance between them by FRET
• Build distance map in 3D for multiple pairs of
  labelled proteins

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Neutron scattering

• Label ribosomes with 15N amino acids to give
  heavy ribosomes (non-radioactive)
• Purify proteins and incorporate into ribosomes
  where all other proteins are light (grown in
  14N amino acids)
• Use reassembled ribosomes in neutron scattering
  experiments to generate distances between heavy
  proteins
• Build distance map in 3D for multiple pairs of
  labelled proteins

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Neutron scattering distance map of 30S subunit
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Chemical crosslinking

• Chemical crosslinking was extensively used for
  identification of close interactions
• Can identify protein-protein or protein-rRNA or
  protein-tRNA or protein-small molecule
• Distance measured depends on length of
  cross-linking reagent.
• UV Light is a zero length crosslinker
• Then must isolate the cross linked components and
  characterise cross linked sites
• Difficult technique to undertake, but yields some
  important results

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Study of the ribosome structure - III

• Purification and characterisation of individual
  proteins and rRNAs
• Cloning of genes for rRNA and proteins
• Characterisation of 2D structures and homologies
  for rRNA
• Identification of homologies for protein sequences

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Purification and characterisation of individual
proteins and rRNAs

• Initial characterisation through purification and
  sequencing of rRNA and rProteins
• Use of 2D gels and microsequencing of proteins
• RNA sequences through T1 fingerprinting and
  chemical sequencing
• These methods were very laborious and slow
• Most work done on E.coli and rat

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Cloning of genes for rRNA and proteins

• Later all components cloned and sequenced, from
  E.coli, humans and other species
• Now very large numbers of genes identified in
  genome projects
• Provides extensive understanding of conserved
  sequences which can be related to function

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Characterisation of 2D structures and homologies
for rRNA

• Sequences allow for computer prediction of 2D
  structures
• Experimental evidence comes from nuclease
  protection experiments, chemical cross linking
  and comparison of sequences from many species
• Overall allowed the construction of a model for
  2D structure and some 3D interactions

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2D structure model for E.coli 16S rRNA

• Predicts 4 domains
• Can map
• Interactions sites in rRNA
• Protein binding sites
• Chemical modification sites

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Homologies between species

• Study of sequences and predicted 2D structures of
  rRNA from many species how significant
  similarities
• Overall 2D structures predicted to be very
  similar, often with limited similarity of
  sequence
• Core of rRNA is similar, with differences located
  around the edges
• Get similar overall results from 16S and 23S
  rRNAs and their eukaryotic homologs (18S and 28S)

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Overall similarities of 2D structures predicted
for 16S rRNA from different phyla
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Study of the ribosome structure - IV

• NMR and X ray crystallography of proteins and
  rRNA components
• X ray crystallography of whole subunits
• Characterisation of binding sites for tRNA and
  antibiotics
• Identification of functional sites and catalytic
  mechanisms
• Development of novel antibiotics

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NMR and X ray crystallography of proteins and
rRNA components

• Early studies on isolated components provided
  structures for individual proteins and regions of
  rRNA
• rRNA structures were mostly short double helical
  regions
• rProtein structures defined a set of common
  motifs for RNA binding, and showed a common
  evolutionary origin for many of the rProteins

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Structure of the RNA binding fold from L7/12 and
L30
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High resolution structures of whole ribosome
subunits

• Two approaches used Cryo electron microscopy and
  X ray crystallography
• Cryo EM involves single ribosome studies with
  novel freeze etching methods. Results in medium
  resolution structures with indications of
  molecular structure

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7Å Structure of 50S subunit
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X ray crystallography of whole subunits

• Limiting factors were crystal quality and data
  complexity
• Crystal quality issues solved by studying
  different species and identifying high quality
  crystals
• Data complexity issues have become less of a
  problem with the develop of high power tuneable X
  ray sources and more powerful computers

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Results of crystal structure validate previous
studies

• Overall structures and models determined by
  previous research proved to be broadly correct
• Overall structure and placement of proteins and
  rRNA was correct
• But details of internal structures, binding and
  catalytic sites was not clear

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3D view of 50S subunit of Haloarcula marismortui
ribosome
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50S subunit showing 23S rRNA
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Conservation of structures in the large subunit

• Red regions are conserved in 95 of species
• Grey regions are not conserved
• Green shows regions which are expanded by
  insertions in eukaryotes to create enlarged
  structures

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Binding sites for antibiotics

• Tetracycline - blue
• Hygromycin B - red
• Pactamycin - green
• All binding to the 30S subunit

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Puromycin binding site is in the catalytic site
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Identification of functional sites and catalytic
mechanisms

• Atomic structure provides a precise picture of
  the ribosome structure and detailed information
  about binding sites for mRNA, tRNA and
  antibiotics
• Movement of tRNA and mRNA through the ribosome is
  better understood
• The catalytic site and mechanisms are better
  defined, although catalytic intermediates are not
  yet characterised

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Development of novel antibiotics

• The characterisation of binding sites for
  antibiotics will assist in understanding their
  mechanism of action
• These data, together with the position of
  mutations which cause antibiotic resistance,
  provides a direction for design of novel
  variations in antibiotic structure
• The structure of the ribosome will provide the
  information required for the development of new
  and specific classes of antibiotics binding to
  new sites and acting through novel mechanisms