Translation &

Proteins
(KCSP 15)

Genetics 244

Introductory Molecular Biology

Departement Genetika
•
Genetics Department
 Translation and
proteins

• What is translation?
Genetics 244 – Translation and proteins

• “Biological polimerisation of amino acids

in polypeptide chains in association with
ribosomes”
• Q&A – tRNA
• Anticodon

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 Ribosomes
• abundant in cell (10 000 copies (eukaryotes have more);
250 nm diameter
• large & small subunits (monosome)
Genetics 244 – Translation and proteins

• rRNA (catalytic function) and proteins (fine-tuning)

• Prokaryotes (E. coli):
• 70S (50S + 30S)
• Large: 23S + 5S + 31 proteins
• Small: 16S + 21 proteins
• Eukaryotes (mammals):
• 80S (60S + 40S)
• Large: 28S + 5.8S + 5S + 50 proteins
• Small: 18S + 35 proteins
• polycistronic transcription of rRNA (prok & euk)

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 Components of pro- and
eukaryotic ribosomes
Genetics 244 – Translation and proteins

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 tRNA
• Small (75-90 nt’s), stable and practically
identical in prok/euk
• Transcribed from larger precursor; spliced
Genetics 244 – Translation and proteins

into 4S tRNA molecules

• Holley et al. 1965 establish complete
nucleotide sequence of tRNAala
• Contains unique bases (posttranscriptional
modifications)
• E.g. inosinic acid; ribothymidylic acid and
pseudouridylic acid (Fig. 15.2)

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 tRNA structure (2-
dimensional)
• Holley et al: 2-dimensional
cloverleaf model
Genetics 244 – Translation and proteins

• Paired stems and unpaired

loops
• 4 large and 1 small stem:
• acceptor-stem with amino acid
binding site (pCpCpA-3’)
• Anticodon stem with 7nt loop
(central 3 = anticodon)
• D-stem and Tψ GC-stem
• Small extra stem – variation
• 5’-end:…pG-5’
• lengths of stems and loops very
conserved; therefore anticodon
loop is always found on same
location on cloverleaf structure.
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 tRNA structure (3-
dimensional)
Genetics 244 – Translation and proteins

• Rich et al.
3-dimensional
L-shaped
structure

• Anticodon loop
and amino acid
binding sites on
opposite
legs of L
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 Charging tRNA

• tRNA in free or charged conformation

Genetics 244 – Translation and proteins

• Aminoacyl tRNA synthetases control

charging of tRNA
• 20 different aminoacyl tRNA synthetases
transfer amino acids to 3’-A residues on
tRNA’s.
• Anticodon: 3’-5’ conformation
• “wobble hypothesis” – 32 tRNA’s

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 Charging tRNA: the
process

• Amino acid + ATP =

Genetics 244 – Translation and proteins

aminoacyladenylic
acid (activated)
• Association with
aminoacyl tRNA
synthetase
• Complex reacts with
a specific tRNA to
transfer AA

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 Translation
Consists of various steps which can be divided into 3 basic
processes: Initiation, elongation and termination.
Genetics 244 – Translation and proteins

Components:

• Large and small ribosomal

subunits
• mRNA
• Charged tRNA’s
• GTP
• Mg++
• Initiation factors (IF1, 2, 3)
• Elongation factors (EF-Tu,
EF-G)
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 Initiation
• Small subunit binds to several IF’s
• Region on the 16S rRNA of small
ribosomal subunit binds to mRNA
Genetics 244 – Translation and proteins

Shine-Dalgarno sequence (AGGAGG

before AUG)
• IF2 binds f-met to small ribosomal
subunit complex and thus sets
reading frame
• Above mentioned “initiation
complex” binds with large ribosomal
subunit
• GTP is hydrolyzed – provides energy
• IF’s released again
• As the two ribosomal subunits bind,
two binding sites for charged tRNA
molecules are formed (P=peptidyl
and A=aminoacyl)

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 The translation process:
Elongation
• Initiation tRNA binds to
P site
Genetics 244 – Translation and proteins

• 2nd tRNA binds to A

site; peptidyl
transferase catalyses
the establishment of a
peptide bond between
the two amino acids
• Covalent bond between
tRNA and amino acid
breaks and uncharged
tRNA is released from P
site – 1st cycle
completed
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 The translation process:
Elongation
• Ribosomal complex shifts three bases to the right (direction of P)
• Third charged tRNA moves into A site
Genetics 244 – Translation and proteins

• Process requires participation of several EF’s

• Energy through hydrolysis of GTP
• Process repeats itself to form peptide chain
• Rate = 15 aa’s/sec; error rate 10-4

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 The translation process:
Termination
• Termination codon
(UAG, UAA, UGA) =
Genetics 244 – Translation and proteins

signal for GTP-

dependent release
factors which cleave
polypeptide chain from
tRNA
• Ribosomal complex
dissociates into
subunits
• Polypeptide folds into
tertiary protein
structure
• Polysomes (Fig. 15.9)
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 Translation in Eukaryotes
• Ribosomes larger
• More complex protein factors - 9 initiation factors
• Transcription and translation spatially and temporaly
Genetics 244 – Translation and proteins

seperated
• mRNA not degraded as fast
• Ribosomes associated with ER
• Initiation tRNA = tRNAiMet (Met-tRNAi) instead of f-met
• 5’-end of mRNA is capped
• Met-tRNAi binds GTP and eIF2
• Complex binds to small subunit
• Complex needs elF4 to bind to 5’-cap of mRNA
• Complex “scans” RNA sequence to find initiation codon (Kozak
sequence – ACCAUGG)
• Anticodon base pairs with initiation codon
• Large subunit binds
• Initiation factors released; GTP hydrolized for energy
• Met-tRNAi in P site, 2nd codon in A
• Elongation and termination very similar (names of protein 15
 Proteins - early
studies
• Garrod & Bateson (1903) demonstrate that pathogens are not the cause of all
diseases, and that some diseases can have a genetic origin (exemplified by
metabolic defects that manifest in the phenotype).
Genetics 244 – Translation and proteins

Propose a relationship between genes (unit factors) and enzymes 16

(ferments).
 Proteins - early
studies
• Experiments by Beadle & Ephrussi (1933) and Beadle &
Tatum (1940) on Drosophila eye pigment and Neurospora
Genetics 244 – Translation and proteins

mutants respectively (Fig 15.11) set the foundations for the

one gene, one enzyme hypothesis.
• Methods used for Neurospora study later used to investigate
metabolic paths in various other organisms as well.
• Refinement of one gene, one enzyme hypothesis leads to
one gene, one protein …and later to one gene, one
polypeptide hypothesis.
• One gene, one polypeptide hypothesis resulted from a
study on sickle cell anemia, which is caused by a hemoglobin
mutation.

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 Genetics 244 – Translation and proteins

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 Protein
structure

• Polypeptide vs. protein

Genetics 244 – Translation and proteins

• Linear nonbranched molecule

• Amino acid: NH2 group, COOH group and R
group
• R provides identity - 20 different kinds
• 4 classes:
• nonpolar (hydrophobic) / polar (hydrophilic)
• Negatively charged / positively charged Fig
15.16
• Enormous number of combinations possible
• Peptide bond covalently binds two
amino acids
(di-, tri-, polypeptides)
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 Genetics 244 – Translation and proteins

acids
Chemical structures of amino

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 Protein structure: 4
Levels
• I° amino acid sequence of linear polypeptide
• II° loose association of amino acids in certain areas of
Genetics 244 – Translation and proteins

polypeptide. Two possible configurations:

Right handed α heliks and pleated β sheet (Fig.
15.18)
• III° 3-dimensional structure (whole polypeptide)
• Specific characteristics that stabilize III° structure:
• Covalent disulfide bonds between closely aligned cysteine residues.
• Nearly all hydrophilic R-groups on outer surface of molecule.
• Nearly all hydrophobic R-groups on inside of molecule.
• IV° only relevant where proteins are composed of more
than one polypeptide chain, e.g. hemoglobin
(Fig. 15.20)

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 Genetics 244 – Translation and proteins

structure
Secondary

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 Genetics 244 – Translation and proteins

Tersiary structure: Myoglobin

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 Genetics 244 – Translation and proteins

Hemoglobin
Quaternary structure:

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 Posttranslational
Modification
• Intricate mechanisms
• Examples:
Genetics 244 – Translation and proteins

• N- and C- termini removed or modified.

• Individual amino acid residues modified (e.g. phosphorylation by kinases).
• Attachment of carbohydrate chains – glycoproteins.
• Polypeptide chain trimmed (e.g. insulin precursor).
• Signal sequences removed (e.g. sequences that determine the delivery of a
protein to a specific destination in the cell where it can perform its duty).
• Complexes with metals (e.g. hemoglobin + 4 Fe ).
++

• Autocatalytic cleavage of precursor protein (viruses).

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 Protein
functions
• Proteins are very abundant in cells, therefore they have many
tasks to perform.
Genetics 244 – Translation and proteins

Specific tasks:
• Very specialized, e.g. Transport of O2 by hemoglobin and
myoglobin
• Collagen and keratin: Structural proteins (connective tissue,
hair and nails)
• Actin and myosin are contractile proteins of muscle tissue.
• Immunoglobulins which perform a protective function in the
immune system of vertebrae.
• Transport of molecules over membranes.
• Regulation of chemical activities by hormones/receptors
• Histones bind DNA in eukaryotes
• Most proteins are enzymes however, and catabolically or
anabolically catalize reactions.
• *Collagen
*Collagen – self study
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 Protein
domains
• Functional areas (50-300 aa’s)
• Unique, stable conformations
Genetics 244 – Translation and proteins

• Often made-up of α -helices and β -sheets

• E.g. catalytic domains and DNA-binding domains
• “exon shuffling” - Gilbert

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