Chapter 14 Protein Synthesis

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Chapter 14 Protein Synthesis
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Some key words in this chapter
Ribosome (???) Codon (???) Anticodon
(????) Reading frame (????) Synthetase
(???) Signal peptide (???)
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14.1 The genetic code

• (1). Codons - three letter genetic code
  (nonoverlapping)
• (2). tRNA - adapters between mRNA and proteins
• (3). Reading frame - each potential starting
  point for interpreting the 3 letter code

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1). Overlapping vs nonoverlapping reading of the
three-letter code
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2). Three reading frames of mRNA

• Translation of the correct message requires
  selection of the correct reading frame

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(4). Standard genetic code
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Features of the genetic code
1). The genetic code is unambiguous. In any
organism each codon corresponds to only one amino
acid. 2). There are multiple codons for most
amino acids(code is degenerate), and synonymous
codons specify the same amino acid 3). The first
two nucleotides of a codon are often enough to
specify a given amino acid
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• 4). Codons with similar sequences specify similar
  amino acids
• 5). Only 61 of the 64 codons specify amino acids
• Termination (stop codons) UAA, UGA, UAG
• Initiation codon - Methionine codon (AUG) also
  specifies initiation site for protein synthesis

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14.2 Transfer RNA
(1). Three-dimensional structure of tRNA

• Transfer RNA molecules are the interpreters of
  the genetic code
• Every cell must contain at least 20 tRNA (one for
  every amino acid)
• Each tRNA must recognize at least one codon
• tRNAs have a cloverleaf type secondary
  structure with several loops or arms

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Cloverleaf secondary structure of tRNA

• Figure next slide
• Watson-Crick base pairing (dashed lines)
• tRNA has an acceptor stem and four arms
• Conserved bases (gray)

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• Cloverleaf structure of tRNA

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(2). Tertiary structure of tRNA
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tRNA arms

• Acceptor stem - amino acid becomes covalently
  attached to tRNA at the 3 end of this stem
• Anticodon arm - contains the anticodon, a
  three-base sequence that binds to a complementary
  codon in mRNA

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• T?C arm - contains thymidylate (T) and
  pseudouridylate (y) followed by C
• D arm - contains dihydrouridylate (D)
• Variable arm - ranges from 3-21 nucleotides

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• Structure of tRNAPhe from yeast

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(3). tRNA anticodons base-pair with mRNA codons

• tRNA molecules are named for the amino acid that
  they carry (e.g. tRNAPhe)
• Base pairing between codon and anticodon is
  governed by rules of Watson-Crick (A-U, G-C)
• However, the 5 anticodon position has some
  flexibility in base pairing (the wobble
  position)

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• Inosinate (I) base pairs
• Inosinate often found at 5 wobble position
• I can form H bonds with A, C, or U
• Anticodon with I can recognize more than one
  synonymous codon

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(4). Codon-anticodon recognition

• Wobble allows some tRNA molecules to recognize
  more than one codon
• Isoacceptor tRNA molecules - different tRNA
  molecules that bind the same amino acids
• Isoacceptor tRNAs identified by Roman numerals or
  codons tRNAIAla, tRNAIIAla or tRNAGCGAla
• Bacteria have 30-60 different tRNAs, eukaryotes
  have up to 80 different tRNAs

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Base pairing at the wobble position
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14.3 Aminoacyl-tRNA synthetases
(???)

• Aminoacyl-tRNA - amino acids are covalently
  attached to the 3 end of each tRNA molecule
  (named as alanyl-tRNAAla)
• Aminoacyl-tRNA synthetases catalyze reactions
• Most species have at least 20 different
  aminoacyl-tRNA synthetases (1 per amino acid)
• Each synthetase specific for a particular amino
  acid, but may recognize isoacceptor tRNAs

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(1). The Aminoacyl-tRNA synthetase reaction

• Aminoacyl-tRNAs are high-energy molecules (the
  amino acid has been activated)
• The activation of an amino acid by aminoacyl-tRNA
  synthetase requires ATP

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(2). Specificity of aminoacyl-tRNAsynthetase

• Attachment of the correct amino acid to the
  corresponding tRNA is a critical step
• Synthetase binds ATP and the correct amino acid
  (based on size, charge, hydrophobicity)
• Synthetase then selectively binds specific tRNA
  molecule based on structural features
• Synthetase may recognize the anticodon as well as
  the acceptor stem

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Structure of E. coli tRNAGln bound to the
synthetase
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(3). Proofreading activity of aminoacyl-tRNA
synthetases

• Some aa-tRNA synthetases can proofread
• Isoleucyl-tRNA synthetase may bind valine instead
  of isoleucine and incorporate it into
  valyl-adenylate
• The valyl-adenylate is usually then hydrolyzed to
  valine and AMP so that valyl-tRNAIle does not
  form

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Model of substrate-binding site in isoleucyl-tRNA
synthetase

• Ile-tRNA binds to Ile about 100x better than Val
  even though they have similar size and charge

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14.4 Ribosomes
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(1). Ribosomes are composed of both rRNA and
protein

• All ribosomes contain two subunits of unequal
  size
• E. coli 70S composed of a 30S and a 50S
• Eukaryotes 80S composed of a 40S and a 60S

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Comparison of prokaryotic and eukaryotic ribosomes
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• Assembly of the 30S ribosomal subunit and
  maturation of the 16S rRNA (E. coli)
• Ribosomal proteins (6-7) bind to 16S rRNA as it
  is being transcribed forming a 21S particle
• Processing and binding of other ribosomal
  proteins completes the mature 30S subunit

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Structure of the 30S ribosomal subunit (T.
thermophilus)
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(2). Ribosomes contain two aminoacyl-tRNA binding
sites

• Ribosome must align two charged tRNA molecules so
  that anticodons interact with correct codons of
  mRNA
• Aminoacylated ends of the tRNAs are positioned at
  the site of peptide bond formation
• Ribosome must hold both mRNA and growing
  polypeptide chain

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Sites for tRNA binding in ribosomes
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14.5 Initiation of translation

• The translation complex is assembled at the
  beginning of the mRNA coding sequence
• Complex consists of Ribosomal subunits mRNA
  template to be translated Initiator tRNA
  molecule Protein initiation factors

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(1). Initiator tRNA

• First codon translated is usually AUG
• Each cell contains at least two methionyl-tRNAMet
  molecules which recognize AUG
• The initiator tRNA recognizes initiation codons
• Second tRNAMet recognizes only internal AUG
• Bacteria N-formylmethionyl-tRNAfMet
• Eukaryotes methionyl-tRNAiMet

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Structure of fMet-tRNAfMet
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(2). Initiation complexes assemble only at
initiation codons

• Ribosome must recognize protein synthesis start
• In prokaryotes, the 30S ribosome binds to a
  region of the mRNA (Shine-Dalgarno sequence)
  upstream of the initiation sequence
• S-D sequence also binds to a complementary base
  sequence at the 3 end of the 16S rRNA
• Double-stranded RNA structure binds mRNA to the
  ribosome

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1). Shine-Dalgarno sequences in E. coli mRNA

• Ribosome-binding sites at the 5 end of mRNA for
  several E. coli proteins
• S-D sequences (red) occur immediately upstream of
  initiation codons (blue)

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2). Complementary base pairing of S-D sequence
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(3). Initiation factors help form initiation
complex

• Initiation factors are required to form a complex
• Prokaryote factors IF-1, IF-2, IF-3
• Eukaryote factors eIFs (8 or more factors)

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Formation of the prokaryotic 70S initiation factor
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(cont)
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(4). Translation initiation in eukaryotes
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14.6 Chain elongation is a three-step microcycle

• The initiator tRNA is in the P site
• Site A is ready to receive an aminoacyl-tRNA
• Elongation is a three-step cycle (1)
  Positioning the correct aa-tRNA in site A
  (2) Formation of a peptide bond (3) Shifting
  mRNA by one codon

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Coupled transcription and translation in bacteria

• Gene is being transcribed left to right
• Ribosomes bind to 5 end of mRNA

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(1). Elongation factors dock an aminoacyl-tRNA
in the A site

• Bacterial elongation factor EF-Tu helps the
  correct aa-tRNA insert into site A
• An EF-Tu-GTP complex binds to all aa-tRNA
  molecules except fMet-tRNAfMet (initiator)
• A ternary complex of EF-Tu-GTP-aa-tRNA binds in
  the ribosomal A site
• If the anticodon of the aa-tRNA correctly base
  pairs with the mRNA codon, complex is stabilized

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EF-Tu binds tRNAs

• EF-Tu binds to acceptor end of aminoacylated tRNA
  (Phe-tRNAPhe)
• Phe residue (green)

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Insertion of aa-tRNA by EF-Tu during chain
elongation
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cont
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Cycling of EF-Tu-GTP
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(cont)
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(2). Peptidyl transferase catalyzes peptide bond
formation

• Peptidyl transferase activity is contained within
  the large ribosomal subunit
• Substrate binding site in 23S rRNA and 50S
  ribosomal proteins
• Catalytic activity from 23S rRNA (an
  RNA-catalyzed reaction)

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• Formation of a peptide bond

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(cont)
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(3). Translocation moves the ribosome by one codon

• Translocation step the new peptidyl-tRNA is
  moved from the A site to the P site, while the
  mRNA shifts by one codon
• The deaminoacylated tRNA has shifted from the P
  site to the E site (exit site)
• Binding of EF-G-GTP to the ribosome completes
  translocation of peptidyl-tRNA

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• Translocation during protein synthesis in
  prokaryotes

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(cont)
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Formation of the peptide chain

• Growing peptide chain extends from the
  peptidyl-tRNA (P site) through a tunnel in the
  50S subunit
• Newly synthesized polypeptide does not begin to
  fold until it emerges from the tunnel
• Elongation in eukaryotes is similar to E. coli
  EF-1a - docks the aa-tRNA into A site EF-1ß -
  recycles EF-1a EF-2 - carries out
  translocation

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14.7 Termination of translation

• E. coli release factors RF-1, RF-2, RF-3
• Translocation positions one of three termination
  codons in A site UGA, UAG, UAA
• No tRNA molecules recognize these codons and
  protein synthesis stalls
• One of the release factors binds and causes
  hydrolysis of the peptidyl-tRNA to release the
  polypeptide chain

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14.8 Protein synthesis is energetically
expensive
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Some antibiotics inhibit protein synthesis

• Some antibiotics prevent bacterial growth by
  inhibiting the formation of peptide bonds
• Puromycin (next slide) resembles the 3 end of an
  aminoacyl-tRNA, and can enter the A site of a
  ribosome
• The peptidyl-puromycin formed is bound weakly in
  the A site and dissociates terminating protein
  synthesis

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14.9 Regulation of protein synthesis
(1). Ribosomal protein synthesis is coupled to
ribosome assembly in E. coli

• Synthesis of ribosomal proteins is tightly
  regulated at the level of translation
• Ribosomal protein genes encode one ribosomal
  protein that inhibits translation of its own
  polycistrionic mRNA by binding near the
  initiation codon of the mRNA

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Comparison of proposed secondary structures of
S7-binding sites (a) S7 site on 16S rRNA (b) S7
site on the str mRNA S7 protein inhibits
translation by binding to the str mRNA molecule
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(2). Globin synthesis depends on heme
availability

• Hemoglobin synthesis requires globin chains and
  heme in stoichiometric amounts
• Globin synthesis is controlled by regulation of
  translation initiation
• Heme-controlled inhibitor (HCI) phosphorylates
  factor eIF-2 which then cannot participate in
  translation initiation
• High heme levels interfere with HCI so that
  globin synthesis proceeds

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• Inhibition of protein synthesis by
  phosphoryl-ation of eIF-2

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(3). The E. coli trp operon is regulated by
repression and attenuation

• The trp operon in E. coli encodes the proteins
  necessary for tryptophan biosynthesis
• Because tryptophan is a negative regulator of its
  own biosynthesis, synthesis can be repressed when
  exogenous Trp is available
• Tryptophan is a corepressor of the trpO operator
  (next two slides)

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Repression of the E. coli trp operon
(continued next slide)
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(continued)
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Attenuation in E. coli

• A second mechanism for regulation of the E. coli
  trp operon depends on translation
• Determines whether transcription of the operon
  proceeds or terminates prematurely
• GC-rich regions in the mRNA trp leader region can
  base pair to form two alternative hairpin
  structures which affect transcription

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(a) Attenuation mechanism for regulation

• mRNA transcript of the trp leader region contains
  four GC-rich sequences which can base-pair to
  form one of two alternative structures

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(b)

• Structure (b) is a pause transcription site

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(c)

• Structure (c) is a more stable hairpin than (b)

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14.10 Posttranslational processing

• Posttranslational modifications can occur either
  before the polypeptide chain is complete
  (cotranslational) or after (posttranslational) De
  formylation of N-terminal residue (prok) Removal
  of N-terminal methionine residue Formation of
  disulfide bonds Cleavage by proteinases Phosphor
  ylation or acetylation

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• Secretory pathway in eukaryotic cells
• Proteins synthesized in the cytosol are
  transported into the lumen of the endoplasmic
  reticulum (ER)
• After further modification in the Golgi, the
  proteins are secreted

(continued next slide)
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(cont)
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(1). The signal hypothesis

• Secreted proteins are synthesized by ribosomes on
  the surface of the endoplasmic reticulum
• A signal peptide is present on the N-terminus
  that signals the protein to cross a membrane
• Signal peptides are 16-30 residues long, and
  include 4-15 hydrophobic residues

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Signal peptides from secreted proteins

• Hydrophobic residues in blue, arrows mark sites
  where signal peptide is cleaved from the precursor

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• Translocation of eukaryotic proteins into the
  lumen of the endoplasmic reticulum

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(cont)
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(2). Glycosylation of proteins

• Many integral membrane and secretory proteins
  contain covalently bound oligosaccharide chains
• Carbohydrate may be from 1 to 80 of the mass of
  the glycoprotein
• A common glycosylation reaction is the covalent
  attachment of a complex oligosaccharide to the
  side chain of an asparagine residue

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Structure of a complex oligosaccharide linked to
an asparagine residue

• Man mannose, Glc glucose, GlcNAc
  N-acetylglucosamine