Transcript No Slide Title

Protein Metabolism
Chapter 27
Protein Synthesis: an overview
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Proteins are end products
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Production must match cellular needs
Must be targeted
Must be degraded when no longer needed
Protein Synthesis is well understood
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Complex biosynthetic pathway
Has been a major emphasis in
biochemistry
Critical importance in every cell
Protein Synthesis: an overview
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Translation
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mRNA-directed biosynthesis of
polypeptides
Requires about 300 macromolecules
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70 ribosomal proteins
20+ enzymes to activate amino acid precursors
12+ enzymes for initiation, elongation,
termination
100+ enzymes for final processing
40+ kinds of tRNA and rRNA
Protein Synthesis: an overview
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Translation
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Requires accurate linkage of 20 AA
In order specified by mRNA
Uses up to 90% of cell biosynthetic energy
Occurs at a high rate of speed
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100 residue polypeptide takes about 5 sec
Must coordinate with targeting and degradation
The Genetic Code: 1950’s
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Where proteins are synthesized:
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Paul Zamecnik and collegues
Determined ribosomes were the site of
protein synthesis
Activation of amino acids:
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Mahlon Hoagland and Zamecnik
Formation of aminoacyl-tRNAs
Catalyzed by aminoacyl-tRNA synthetases
The Genetic Code: 1950’s
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Crick’s adaptor hypothesis
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Explained how the 4-letter language of
nucleic acids was translated into the 20letter language of proteins
Proposed that it was a small nucleic
acid
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Part bonds to a specific amino acid
part recognizes the codon
The Genetic Code: 1960’s
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Determination of the triplet character
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Francis Crick and Sidney Brenner: 1961
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Induced mutations in bacteriophage T4
Formulated the following hypotheses:
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The code is read in sequence from a fixed point
The code is comma-free
The code is a triplet code
 Codon: group of bases specifying an AA
 Triplet bases=codon
The code is degenerate
The Genetic Code: 1960’s
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Francis Crick and Sidney Brenner: 1961
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Used frameshift mutations
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Observed that insertions or deletions shifted
the frame
Reading frame: established by the first
codon
Did not absolutely prove the triplet code
The Genetic Code: 1960’s
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Genes are colinear with their specified
polypeptides
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Charles Yanofskey
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Used E.coli mutants for tryptophan synthase
Specified by the trpA gene
Mutations in the gene corresponded to changes
in the protein
The Genetic Code: 1960’s
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What were the 3 base codons for each
amino acid?
The Genetic Code: 1960’s
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Deciphering the Code
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Marshall Nirenberg and Heinrich Matthaei:
1961
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Revealed the base composition of codons, but
not the base sequence
The Genetic Code: 1960’s
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Deciphering the Code
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Nirenberg and Philip Leder: 1964
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Determined about 50 of the 64 possible triplet
codons
The Genetic Code: 1960’s
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Deciphering the Code
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H. Gobind Khorana: 1964
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Completed the dictionary
Reconfirmed the triplet code
Confirmed I.D. of many codons
Filled out missing portions of the genetic code
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61 of 64 codons specify AA
Other 3: termination codons
The Genetic Code: 1960’s
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Meanings for all codons established by
1966
Cracking the genetic code is regarded
as one of the most significant scientific
discoveries of the twentieth century
Nature of the Code
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The code is DEGENERATE
Does not mean that the code is
inaccurate or imperfect
Nature of the Code
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The code is DEGENERATE
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AA may be specified by more than one
codon:
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AA specified by 6 codons: Arg, Leu, Ser
AA specified by 4,3 or 2 codons: most
AA specified by 1 codon:
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Methionine (AUG)
Tryptophane (UGG)
Nature of the Code
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The code is DEGENERATE
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Synonyms: codons that specify the same
AA
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Initiation (start) codon: AUG
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Codons usually differ only in the 3rd base
GUG also: or codes for Val
AUG also codes for Methionine
Stop codons (Nonsense codons): UAA,
UAG, UGA
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Do not code for an AA
Nature of the Code
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The arrangement of the code is
nonrandom
Nature of the Code
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The reading frame is correctly set in the
beginning of the mRNA readout
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Initiation Codon: signals start
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AUG: in prokaryotes and eukaryotes
Codes for Met if occurs internally
Reading frame moves from one triplet to
the next
Nature of the Code
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The reading frame is correctly set in the
beginning of the mRNA readout
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Termination codons (aka: STOP codons;
nonsense codons)
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Signal end of protein synthesis
Found first in E. coli as single base mutations
Called UAG: amber; UAA: ochre; UGA: opal
Eg: an amber mutation changes another codon
into UAG
Nature of the Code
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The reading frame is correctly set in the
beginning of the mRNA readout
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Open Reading Frame
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Sequence of codons (50 or more) without a
termination codon
Long ones usually correspond to genes for
proteins
Eg: protein with MW of 60,00=open reading
frame of ~500 codons
Nature of the Code
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The reading frame is correctly set in the
beginning of the mRNA readout
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A mistake of a single base will put all
subsequent codons out of register
Nature of the Code
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Some viral DNA segments contain
overlapping genes in different reading
frames
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Any nucleotide sequence has 3 potential
reading frames
Nature of the Code
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Some viral DNA segments contain
overlapping genes in different reading
frames
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Any nucleotide sequence has 3 potential
reading frames
In theory: same sequence could code for 2
or 3 different polypeptides
Most DNA sequences: one reading frame;
one protein product
Nature of the Code
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Some viral DNA segments contain
overlapping genes in different reading
frames
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Frederick Sanger: 1976
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DNA of bacteriophage 0X174
Found genes within genes
Bacteria also show such coding economy
Overlapping genes: in small, single
stranded DNA phages
Nature of the Code
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The genetic code is nearly universal
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A basic concept
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One organism can translate genes of another
Eg: E. coli can translate human genes
Basis of genetic engineering
Suggests common evolutionary ancestor
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Single genetic code
Strong selection against mutation
Nature of the Code
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The genetic code is nearly universal
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Minor Variants have been found
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Mammalian Mitochondria (1981)
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Contain genes
Have variations on the standard genetic code
 AUA=STOP
 UGA=Trp (not STOP)
 AGA, AGG= STOP (not Arg)
Increased degeneracy simplifies code
Nature of the Code
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The genetic code is nearly universal
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Some variants in prokaryotes, protista
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UGA= STOP and selenocysteine (sometimes)
Transfer RNA
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NA do not specifically bind AA
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Question: How do cells translate code of
Base Sequence into language of
polypeptides?
Adaptor Hypothesis of Francis Crick
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Postulated:
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Genetic code read by molecules that recognize
specific codon
That molecule carries corresponding AA
tRNA was Crick’s adaptor molecule
Transfer RNA
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Primary and Secondary structures
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Robert Holley: 1965
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Reported 1st base sequence of a biologically
significant NA
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Took him 7 yrs
Sequenced yeast Alanine tRNA (tRNAAla)
Invented methods to sequence RNA
Had to purify: yeast tRNAAla has 10 modified
bases … made it harder!
Transfer RNA
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Techniques now vastly improved
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Can sequence in a few days
Know sequence for over 300 tRNAs
tRNA: primary structure
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Vary in length
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60-95 nucleotides
Most are ~75 nucleotides
tRNA: secondary structure
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5’ terminal phosphate group
Acceptor (Amino Acid Stem): 7bp stem
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Includes 5’ terminal nucleotide
May contain non-WC base pairs
AA residue: 3’-terminal OH group
tRNA: secondary structure
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D arm (DHU: dihydrouridine arm)
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3-4 bp stem
Ends in loop
Loop often has modified base:
dihydrouridine (D)
Function?
tRNA: secondary structure
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Anticodon Arm
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5 bp stem
Ends in loop
Loop contains anticodon
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3 bases
Complimentary to codon
tRNA: secondary structure
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T arm
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5bp stem
Ends in loop with modified bases
Function?
Termination
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All tRNA terminates in CCA
And a free 3’-OH group
tRNA: secondary structure
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Variable arm
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Greatest variability
3-21 nucleotides
May have stem up to 7 bp
Invariant positions
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Always same base
13
tRNA: secondary structure
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Semivarients
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8
Always a purine, or always a pyrimidine
Mostly in loops
Purine on 3’ side of anticodon: modified
Correlated invarients
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Pairs of non-stem nucleotides
Are base-paired in all tRNA
Tertiary structure
Modified tRNA bases
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Stricking feature of tRNA
Up to 20% modified bases
Over 50 such bases
Functions?
tRNA: tertiary structure
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3-D structure: 1974
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Based on X-ray crystal structure of yeast
tRNAPhen
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Alexander Rich and Sung Hou Kim
Aaron Klug (used different crystal form)
Molecule assumes L-shaped conformation
tRNA: tertiary structure
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One leg
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Acceptor and T-stem
A-DNA like helix
Other leg
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D-stem
Anticodon stem
tRNA: tertiary structure
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Characteristics of L shape
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Each leg ~60 A long
Anticodon and acceptor at opposite ends of
molecule
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~76A apart
20-25 A wide: essential to function
tRNA: tertiary structure
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Maintained by hydrogen bonds
Also stacking interactions
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Similar to proteins
9 bp cross-link the tertiary structure
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Mainstay
All non W-C base pairs but one
Most are invariant, or semivarient
Very compact
Aminoacyl-tRNA synthetases
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Group of enzymes
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Are AA specific
Catalyze attachment of correct AA to
correct tRNA
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AA to 3’ terminal of acceptor stem
Forms aminoacyl-tRNA
2 classes
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Based on structure
Differ in 2nd reaction step
Aminoacyl-tRNA synthetases
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Reaction energy derived from ATP
2 steps
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First: activation of AA
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Formation of enzyme bound intermediate:
aminoacyl-AMP
Second: formation of aminoacyl-tRNA
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Aminoacyl group is tranfered to correct tRNA
Method depends on enzyme class
Aminoacyl-tRNA synthetases
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Overall reaction: irreversible
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AA+tRNA+ATP
Aminoacyl-tRNA + Amp + PPi
Aminoacyl-tRNA synthetases
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Cells have at least one aminoacyl-tRNA
synthetase for each standard AA
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Diverse group
Over 100 characterized
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Each: 4 subunits
Between organisms: same enzyme;
considerable sequence homology
Between enzymes: little similarity
Methods of recognition between enzyme
and AA are idiosyncratic
Aminoacyl-tRNA synthetases
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Proofreading: correct AA to correct
tRNA
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Critical
Occurs at several sites
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When AA binds (activation to aminoacyl-AMP)
Enzyme has a second active site
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Will catalyze removal of incorrect AA
Based on 3-D structure
Third site: sometimes: removes incorrect AA
Aminoacyl-tRNA synthetases
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Proofreading critical
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Esp with structurally similar AA
Second genetic code:
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Interactions between synthetases and
tRNA
Critical to accuracy of protein synthesis
Complex “coding rules”
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Some nucleotides are recognition factors
Codon-Anticodon Interactions
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tRNA is selected by codon-anticodon
interactions
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Called Codon recognition
Aminoacyl group not involved
Variable third position: large part of
code degeneracy
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Isoaccepting tRNAs+different TRNAs
specific for same AA
Codon-Anticodon Interactions
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Many tRNAs can bind to multiple codons
(specify cognate AA)
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Non W-C bp often at 3rd position
The Wobble Hypothesis: accounts for
codon degeneracy
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Developed by Crick
The Wobble Hypothesis
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First two codon –anticodon pairs are
typical W-C: establish structural
constraints
Third codon position
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Called the wobble base
Allows limited conformational adjustments
Non W-C pairs can be accomodated
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Called wobble pairing
The Wobble Hypothesis
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Crick deduced the most likely wobble
pairs
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C or A: only with W-C pairs
U in anticodon: A or G in Codon
G in anticodon: U or C in Codon
U in anticodon: U, C or A in Codon
The Wobble Hypothesis
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Minimum “set of tRNAs for translation
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32 tRNAs
31 to translate 61 coding triplets
Initiation requires separate tRNA
Most cells have >32 tRNAs
If either of first two bases are different on
codons for the same AA: require different
tRNAs (isoaccepting tRNA)
Ribosomes
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History
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Albert Claude (late 1930’s)
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First observed
Used dark field microscopy
Called them microsomes
George Palade (mid 1950’s)
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EM
Established they were not artifacts
Ribosomes
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Name:~2/3 RNA; 1/3 protein
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Microsome: artifactual vesicle, in EM
Ribosome: site of protein synthesis;
abundant in cells that make a lot of protein
Paul Zamecnik (1955)
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Confirmed ribosomes were the site of
protein synthesis
Ribosomes
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Ribosomal protein synthesis: 3 phases
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Chain initiation
Chain elongation
Chain termination
Ribosomes
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Structure of prokaryote ribosome
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Spheroid particle
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70S
~250A dia
Two subunits (discovered by James Watson)
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Small: 30s
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16s rRNA molecule
21 different polypeptides
Large: 50S
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5S rRNA
23S rRNA
32 different polypeptides
Ribosomes
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Structure of prokaryote ribosome
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~20,000 ribosomes in E. coli
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~ 80% of cell RNA
~ 10% of cell protein
Ribosomes
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Have been crystallized recently
3-D studies: Image reconstruction by EM
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Aaron Klug
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Used images from several views
Mathematical reconstruction
Small subunit
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“Mitten”- shaped
Head, base, cleft, platform
Ribosomes
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3-D studies: continued
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Large subunit
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Spheroidal with 3 protuberances
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Central protuberance
Ridge
Stalk
Tunnel
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~25A dia
100-120 A long
Originates in a cleft between protuberances
Ribosomes
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Secondary structure of rRNA
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“flower-like” in the 16S rRNA of 30S subunit
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4 domains: sequenced (1542 nucleotides)
46% base paired
Double-helical areas tend to be short ~8bp
Shape largely determines shape of 30S subunit
Large subunit: rRNA has been sequenced
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5S: 120 nucleotides
23S: 2904 nucleotides
Extensive secondary structure
Ribosomes
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Ribosomal Proteins
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Insoluble: difficult to isolate
Naming conventions
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From small subunit: S
From large subunit: L
Followed by number based on
electrophoretogram: Eg: S20/L26
All 52 E. coli proteins have been sequenced
Little sequence similarity
Most are rich in Lys, Arg
Ribosomes
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Ribosomal subunits are self-assembling
Ribosomal architecture:
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Component positions have been determined
by Immune EM
Verified: eg: neutron diffraction measurement
Functional Components
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Identified by affinity labeling techniques
Ribosomes
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Ribosomal architecture:
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Functional Components
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Binding mRNA on small subunit
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Anticodon binding site
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3’ end of 16S rRNA
Located on “platform”
Binds from “head” to “base”
Small subunit
cleft
GTP reactions
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Large subunit
Stalk (4 L7/L12 subunits)
Ribosomes
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Ribosomal architecture:
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Functional Components: continued
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Peptidyl transferase function
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Ribosome binding to membranes
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Large subunit
Adjacent to exit tunnel
Large subunit
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Catalyzes peptide bond formation
“valley” of small subunit
Main job: biochemical tasks
Also, tRNA binding
Small subunit: binds mRNA and tRNA
Ribosomes
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Eukaryotic Ribosomes
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Resemble prokaryotic
Larger, more complex
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~23 nm dia
Sedimentation coefficient= ~80S
2 subunits
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Large subunit: 60S
Small subunit: 40S
Ribosomes
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Eukaryotic Ribosomes
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Large subunit
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rRNA: 3
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5S
5.8S
28S
49 polypeptides
Small subunit
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rRna: 18S
33 polypeptides