Translation: Building Proteins

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TranslationBuilding Proteins

• 22.228
• Dr. Bill Diehl-Jones

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Agenda

• Functions of proteins
• Protein structure
• Steps in Translation
• 1. activation of amino acids
• 2. initiation
• 3. elongation
• 4. termination
• The endomembrane system

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Some general functions of proteins

• enzymes protein catalysts.
• structural elements e.g. tubulin
• contractile elements e.g. myosin
• control activity of genes e.g. transcription
  factors
• transport material across membranes e.g.
  glucose transporter
• carriers e.g. hemoglobin
• hormones e.g. insulin
• antibodies

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Composition of Proteins

• 1. Amino Acids
• building blocks of proteins.
• organic acids that contain an amino group
• General Structure
• ? carbon first carbon after the carboxyl group
• ? carbon is almost always asymmetric -- leads to
  D and L stereoisomers of amino acids

Amino group
Carboxyl group
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Composition of Proteins

• 2. R groups
• R side chain of variable structure
• any of 20 different groups
• differences in R groups account for different
  properties of amino acids and proteins
• R groups can be broadly classified as
• i. polar charged
• ii. polar uncharged
• iii. Nonpolar
• iv. R groups with unique properties

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Composition of Proteins

• 3. Peptide bond or Amide linkage
• links ?-amino group of one amino acid with
  ?-carboxyl group of adjoining amino acid
• the linkage in dipeptides and in polypeptides

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Composition of Proteins

• 4. Covalent modifications of proteins
  (polypeptides)
• modifications made by additions to the amino acid
  R groups
• an example is placement of a phosphate on serine,
  threonine and tyrosine residues

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Structure of Proteins

• 1. Primary Structure
• the sequence of amino acids in a polypeptide
• most polypeptides contain over 100 amino acids
• in a polypeptide chain, amino acids are termed
  residues
• N-terminus is the end of a polypeptide with a
  free ? amino group
• C-terminus is the end of a polypeptide with a
  free ? carboxyl group.

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Structure of Proteins

• 2. Protein conformation
• three dimensional structure of a protein
• secondary, tertiary and quaternary structure
  describes conformation
• primary structure determines secondary, tertiary
  and quaternary structure of proteins.

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Structure of Proteins

• 3. Secondary structure of protein
• the local spatial arrangement of the atoms in the
  backbone of a polypeptide (fixed configuration of
  the polypeptide backbone)
• results from hydrogen bonding between the oxygen
  of one peptide group and the nitrogen of another
  peptide group (through the H attached to the
  nitrogen)
• R groups are not involved
• secondary structure is limited to a small number
  of
• Conformations
• two common secondary structures

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Secondary Structures

• ?-helix
• cylindrical, twisting spiral
• each amino acid is hydrogen bonded to its fourth
  neighbor on both sides

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Secondary Structures

• Pleated sheets or ?-pleated
  sheets
• polypeptide chains of pleated sheets are
  stretched out and lie side by side, either
  parallel or antiparallel to one another
• bonded groups may be portions of same chain
  folded back on itself or bonded groups may be on
  separate chains.

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Secondary Structures

• Unorganized portions of protein
• 60 of the polypeptide chain in an average
  protein exists as ? helices and ?-pleated sheets
• remainder is in random coils and turns
• Do not get continuous helix or pleated sheet for
  two reasons
• juxtaposition of two bulky or similarly charged
  side chains
• proline helix breaker'

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Depiction of Secondary Structure

• ? helices are represented by helical ribbons
• ? sheets as flattened arrows
• connecting segments as thin strands.

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Protein Motifs

• Substructure found among many different proteins
• Recurring combinations of secondary structure
• Combinations of ? helices, ? sheets and loops
  found in a variety of proteins
• Usually associated with a particular function
• e.g. zinc finger bundle of three secondary
  structures an ? helix and a pair of antiparallel
  ? sheets
• This motif generally is present in proteins that
  bind DNA.

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Tertiary Protein Structure

• The way that regions of secondary structure are
  oriented with respect to each other.
• Tertiary structure predominates in globular
  proteins.
• Monomeric proteins consist of a single
  polypeptide chain folded into its tertiary
  structure.
• Tertiary structure results from side chain
  interactions. These are
• hydrogen bonds
• hydrophobic bonds
• ionic bonds
• disulfide bond
• covalent bond between two cysteines

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Some non-covalent bonds involved in protein
structure
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Domains

• Region within a protein that folds and functions
  in a semi-independent manner
• Modules of tertiary structure
• Different domains often represent parts that
  function in a semi-independent manner.
• e.g. bind different factors
• Some polypeptides containing more than 1 domain
  are thought to have arisen during evolution by
  fusion of genes

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Quartenary Structure
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Multi-protein complexes

• Physical association of different proteins, each
  with a specific function, to coordinate a larger
  function
• e.g. pyruvate dehydrogenase complex

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RibosomesOrganelles central in protein
synthesis

• mRNA
• see previous lecture
• Ribosomes
• RNA/protein complexes.
• ribosomes are made up of two different subunits.
• in eukaryotes, these are 60S and 40S and together
  form a functional unit, which is 80S.
• in prokaryotes, these are 50S and 30S and
  together form a functional unit, which is 70S.

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Ribosomal RNA
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tRNA

• Function is to recognize an mRNA codon and to
  position correct amino acid into growing
  polypeptide.
•  Two important sites
• amino acid attachment site at 3 end.
• attachment of amino acid makes tRNA an
  aminoacyl-tRNA (aa-tRNA)
• Anticodon
• aa-tRNA recognizes correct mRNA codon during
  translation through pairing between 3
  complementary bases in mRNA codon and tRNA
  anticodon.

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tRNA
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tRNA
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tRNA
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The Process of Translation
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Step 1. Activation of Amino Acids

• Each amino acid is attached to a suitable tRNA.
•  For each amino acid there is at least one
  specific tRNA.
•  For each amino acid there is a specific
  activating enzyme to attach the amino acid to its
  tRNA.
•  These enzymes are aminoacyl-tRNA synthetases.
•  Product is an aminoacyl-tRNA (aa-tRNA), or
  charged tRNA.
•  
• amino acid ATP E ? aa-AMP-E PPi
• aa-AMP-E tRNA ? aa-tRNA E AMP

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Step 2 Initiation of protein synthesis

• 1. 30S ribosomal subunit binds mRNA at AUG
  initiation codon
• 2. N-formyl-methionine-tRNA hydrogen-bonds to
  initiation codon, AUG.
•  3. 50S ribosomal subunit binds to initiation
  complex to form functional ribosome 
• 4. ribosome has two sites
• a. A (aminoacyl) site - all aminoacyl tRNAs enter
  ribosome-mRNA complex here except for N-formyl
  methionine-tRNA.
• b. P (peptidyl) site - site from which tRNA
  donates amino acids to growing peptide.
•  5. N-formyl-methionine-tRNA enters at P site.

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Elongation CycleThree Major Steps

• 1. incoming aminoacyl-tRNA binds to next codon of
  mRNA in A site.
• 2. peptide bond is formed between amino group of
  newly bound aminoacyl-tRNA and carboxyl group of
  1st aa.
• a. catalyzed by peptidyl transferase
• b. results in "empty" tRNA and elongated
  peptidyl- tRNA
• 3. Translocation
• a. peptidyl-tRNA moves from A site to P site.
• b. ribosome moves along mRNA by one codon.
• c. translocation requires GTP and G factor.
• d. A site is open for next aa-tRNA.

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Polypeptide Chain Termination

• Elongation cycle is repeated until ribosome
  reaches a stop or nonsense codon,
• either UAA, UAG or UGA.
•  Release factors
•  Ester linkage between tRNA and polypeptide is
  cleaved.
• a. empty tRNA is released.
• b. ribosome separates into subunits.
• c. polypeptide is released.
•  Polypeptide has grown from N to C terminus.

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Post-Translational Modification

• Polypeptide is often cleaved.
•  Addition of groups (e.g. phosphate) to R group
  of amino acid residues.
•  Polypeptide becomes properly folded.
•  Folding can occur spontaneously but is assisted
  by chaperones and protein disulfide isomerases

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Glycosylation in the rough endoplasmic reticulum.

• Nearly all proteins made in the RER are
  glycosylated
• includes internal membranes, internal
  compartments, secreted proteins, the plasma
  membrane.
• the function of the glycoproteins includes unique
  recognizability in binding to other proteins
  (e.g.. cell recognition)
• the sequence of carbohydrate units (I.e. sugars)
  is very important in this function
• they are built in a step-wise fashion, sugars
  added one at a time
• based on a series of sequential additions by
  glycosyl transferase enzymes, starting in the RER

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Formation of polyribosomes or polysomes

• A complex of mRNA and many ribosomes
• mRNA read by several ribosomes simultaneously
• Thus several polypeptide chains can be made from
  it simultaneously
•  This greatly increases rate of protein synthesis

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The Endomembrane System
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Three general destinations of proteins

• 1. Remain in cytoplasm
• 2. Sent to cellular compartments via the
  cytoplasm
• Nucleus (by NLS)
• mitochondria, chloroplasts, peroxisomes (other
  a.a. sequence tags)
• Peripheral proteins of the cell membrane
• 3. The endomembrane system via the endoplasmic
  reticulum
• Defined as a functionally and structurally
  interrelated group of membrane-bound cytoplasmic
  organelles

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General Pathways of New Proteins
membrane fusion
lysosome
secretion
mitochondrion
golgi
STARTcytoplasm
R.E.R.
nucleus
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Proteins Destined for the Endomembrane System

• Some proteins made in the cytosol, stay there
• Many proteins are directed to the E.R. for
  PROCESSING, and SORTING
• Destined for residence in or transfer through the
  compartments of the ENDOMEMBRANE SYSTEM.
• Two destinations
• the lumen, or
• the membrane itself

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Outward bound elements of the endomembrane system

• endoplasmic reticulum (ER), with its rough (RER)
  and smooth (SER) components
• Golgi apparatus
• lysosomes
• plant vacuoles
• secretory vesicles and granules which release
  material to the extracellular space by fusing
  with the
• the plasma membrane

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Structure of the E.R.

• Membrane-bound spaces
• The outer membrane of the nuclear envelope is
  studded with ribosomes and is continuous with the
  rough ER.
• Smooth endoplasmic reticulum (SER), no ribosomes,
  specialized enzymes, functions

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Structure of the E.R. contd

• The appearance of the RER and SER vary in
  different cell types.
• RER is very abundant in secretory cells,
  Pancreatic Acinar Cells, and mucous secreting
  cells (4.8.11)
• SER abundant in testicular leydig cells (steroid
  synthesis) (fig4.8.1) and skeletal muscle cells
• Transitional elements (ERGIC) are specialized ER
  cisternae from which membrane vesicles (MV) bud
  off that deliver materials from the ER to the cis
  region of the Golgi apparatus

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Chemical modification of proteins in the E.R.
(continuing to the Golgi)

• 1. disulfide bond formation (cysteines) and
  folding
• the tertiary structure of the final protein
• Protein folding Proteins have a correct or
  native state. the tertiary structure of a
  protein needs to be correct for it to work
  properly. It can be misfolded or denatured
• Molecular Chaperones are proteins that bind to
  and alter the folding of newly forming protein
• quality control proteins that mis-fold cannot
  bind to chaperones are destroyed
• With several cysteines, order is required for
  protein disulfide isomerase to work right.

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Chemical modification of proteins in the E.R.
contd

• 2) Addition of extra chemical groups
• e.g. methyl, acetyl, formyl, sulfate, hydroxyl,
  etc.
• e.g. hydroxylation of lysine and proline residues
  of a forming collagen molecule
• 3) Addition of lipids, lipoproteins are
  lipid/protein molecules of the cell membranes
• includes GPI anchored proteins discussed later.
• 4) Formation of multimer proteins
• - quaternary structure of proteins
• 5) Proteolytic cleavage
• - removal of signal peptides

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Chemical Modifications

• 6) Glycosylation
• Most proteins destined for secretion, the
  extracellular matrix, the endomembrane system and
  the plasma membrane are mostly glycoproteins

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The Process of Glycosylation

• The process of glycosylation, occurs in the RER
  and the golgi
• A step-wise process
• By a series of Glycosyltransferase enzymes
• Substrates 1. the growing chain and 2. a
  sugar-nucleotide molecule such as GTP-Mannose

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Rough endoplasmic reticulum

• Site of protein production, abundant in cells
  such as pancreatic acinar cells and mucous
  secreting cells of the gut. (Karp figs 8.9
  8.11)
• Contains membrane-bound ribosome.
• RER is the site of synthesis of proteins destined
  for
• Secretion and
• Integral membrane proteins and proteins within
  lumen of the endomembrane system
• Endoplasmic reticulum, golgi, lysosomes,
  vesicles, plant vacuoles

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NOT the RER!

• Proteins for the following locations are made
  within the cytoplasm, not the RER.
• Cytosol
• Peripheral proteins of the cell membrane
• Proteins sent to the nucleus
• Proteins destined for chloroplasts, mitochondria,
  peroxisomes

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How do proteins get to the RER?

• Proteins directed to the R.E.R. by direction by a
  SIGNAL SEQUENCE
• - a sequence of 16 - 30 amino acids on the
  primary a.a. structure
• - usually at the N-terminal (first part of the
  protein synthesized)
• - a terminal region of charged amino acids
• - this was the first of a number of different
  signal sequences Blobel, Sabatini, Dobberstein,
  1970s Nobel 1999
• - How it works the protein synthesis starts on a
  cytoplasmic ribosome/mRNA complex
• 1. the signal sequence is the first part of the
  polypeptide made (amino terminal)
• 2. the polypeptide is 80 - 100 aa long before the
  signal is clear of the ribosome

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Getting to the RER (continued)

• 3. while in cytoplasm it is recognized and bound
  to a protein called the Signal Recognition
  Particle (SRP)
• - six to 15 polypeptides complexed to a special
  short 7S RNA
• 4. This causes translation to stop
• just a temporary halt
• translation will resume after it gets to the RER
• 5. SRP, protein, ribosome, mRNA complex bind to
  an SRP receptor on the RER membrane
• 6. the nascent protein goes into a channel in the
  e.r.
• Translocon channel
• partially open channel (15A) opens to 50A.
• it is normally sealed by Bip protein

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Getting to the RER (continued)

• 7. Translation resumes
• - SRP lets go of the signal sequence, insert into
  translocon, converting GTP to GDP, leaves.
• - after a time, the SRP receptor hydrolyzes GTP,
  leaves the translocon
• the time it takes for SRP receptor to hydrolyze
  GTP is a timing mechanism, giving the signal
  peptide a chance to attach to the channel.
• 8. Growing chain within RER undergoes further
  processing. Processing proteins are present in
  either the lumen of the e.r. or the membrane.
• a. Signal peptidase (membrane) removes the signal
  sequence
• b sugar-addition starts in e.r. (by membrane
  bound proteins), as the protein is being made.

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cytosol
Signal 15-30 a.a.
80-100 aa
NH3
2 GDP 2 Pi
SRP
2 GTP
GTP
GTP
BiP
BiP
BiP
Signal peptidase
BiP
opens channel (sealed by BiP)
BiP
two binding sites 1. SRP receptor and
2.translocon channel
NH3
two destinations 1) lumen of RER 2) E.R. membrane
folding
Karp fig. 8.12
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• 10). some proteins get incorporated into the
  membranes
• - hydrophobic transmembrane segments
  Stop-transfer sequence. at least 15 hydrophobic
  aa in a row. co-translational insertion stops,
  the translocon opens (side-door), sends the
  sequence into the membrane
• - then a Start transfer sequence tells the
  translocon that additional amino acids are to be
  added outside the membrane.
• - a series of starts and stops results in a
  protein with several trans-membrane parts.

cytoplasm
lumen of e.r.
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Insertion of membrane proteins into the e.r.
membrane

• Stop-transfer sequence.

Cytosol side
NH3
Lumen of e.r.
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• Multiple stop and start-transfer sequences. Using
  internal start-transfer sequences, protein with
  two or multiple trans-membrane passes. (note this
  diagram could not really work, because the
  protein is being inserted as it is being made, so
  grey part is looped in as it is made.)

Internal start
stop
NH3
The signal peptidase has to stop its activity on
the internal site, otherwise it would cut off the
grey portion!
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Proteins leave the RER to the golgi.
membrane fusion
lysosome
secretion
mitochondrion
- transitional elements of the RER, have no
ribosomes on them. A series of Tubules and
Vesicles called ERGIC, communicate between ER and
Golgi.
golgi
STARTcytoplasm
R.E.R.
nucleus