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From Gene to Protein
Protein Structure and Folding
What is a gene?
• A unit of heredity that is transferred from a parent to
offspring and is held to determine some characteristic
• Proteins coded directly by genes
• A distinct sequence of nucleotides forming part of a
chromosome
“The basic physical unit of heredity; a linear sequence of
nucleotides along a segment of DNA that provides the
coded instructions for synthesis of RNA, which, when
translated into protein, leads to the expression of
hereditary character”.
The 21st and 22nd genetically encoded amino acids
• Selenium is an essential nutrient for many organisms including
humans.
• The major biological form of selenium is a component of the amino
acid selenocysteine.
• Encoded in > 15 genes in prokaryotes that are involved in redox
reactions, and in > 40 genes in eukaryotes that code for various
antioxidants and the type I iodothyronine deiodinase of the thyroid
gland.
• Selenoproteins are essential for mammalian development.
• The UAG codon in some instances can trigger incorporation of
pyrrolysine rather than termination.
• The 22nd amino acid, pyrrolysine, was recently identified in a few
archaebacteria and eubacteria. In the archaebacterium
Methanocarcina barkeri, pyrrolysine has been found in some
methylamine methyltransferases. required to generate methane
from methylamines
Modified Nucleotides and Codon Bias
• The degeneracy of the code results in some
amino acids being coded for by as many as six
codons (e.g. leucine, serine), whereas others
are coded for by as few as one (e.g.
methionine, tryptophan).
• When bases in the anticodon are modified,
further pairing patterns become possible in
addition to those predicted by the regular and
wobble pairing
• Inosine, which is often present in the first
position of the anticodon, can pair with any one
of three bases, U, C, and A.
• In contrast, modification of uracil (U) to 2thiouracil restricts pairing to A alone, because
only one hydrogen bond can form with G.
• Codon bias = the frequencies with which different
codons are used vary significantly between
different organisms and between proteins
expressed at high or low levels within the same
organism.
Example
• Mammalian genes commonly use AGG and
AGA codons for arginine, whereas these are
very rarely used in Escherichia coli.
• E. coli is a bacterium often used for expression
of recombinant human proteins.
• Correlating with this observation, in E. coli, the
tRNAArg that reads the infrequently used AGG
and AGA codons for arginine is present only at
very low levels.
Example 2
• Tetrahymena, the ciliate that played an
important role in the discovery of telomerase,
possesses tRNAs that read the canonical stop
codons UAA and UAG as glutamine (Gln),
making these genes impossible to express
heterologously without some type of redesign
strategy of the gene or host.
Peptide Bonds
Four Levels of Structure
Globular proteins
• The overall shape of most
proteins is roughly
spherical.
• Proteins that adopt this
form are called globular
proteins.
• E.g lysozyme folds up into a
globular tertiary structure,
forming the active site
within a deep pocket
between folded regions.
Fibrous proteins
• Fibrous proteins have properties distinct from globular
proteins.
• A common feature of most fibrous proteins is their long
filamentous or “rod-like” structure.
• A triple helical arrangement of polypeptide chains is
exemplified by the collagen family of proteins, which are a
major structural component of skin, tendons, ligaments,
teeth, and bone.
• The α-keratins, which are structural components of
mammalian hooves, nails, and hair, adopt a structure
composed of “coiled coils” of α-helices.
• A variation on this helical theme is structure of actin
filament.
Size and complexity of proteins
• There is tremendous variation in the size and
complexity of proteins.
• The molecular weight of proteins and the number of
subunits (polypeptide chains) shows much diversity.
• Dalton units (1 Da is equivalent to 1 atomic mass unit)
are used frequently in the protein literature to describe
the molecular weight.
• This is the absolute molecular weight representing the
mass in grams of 1 mole of protein.
• Typical polypeptide chains have molecular weights
ranging from 20 to 70 kDa (20,000–70,000 Da).
Protein function
• Proteins provide the structures that give cells integrity
and shape – cytoskeleton.
• Others serve as hormones to carry signals from one cell
to another, or to transport oxygen around the bodies of
multicellular organisms.
• Of particular significance for molecular biologists are
the proteins that mediate the activities of genes at all
points in the flow of genetic information from
replication to transcription to translation.
• Another vital role of proteins is to serve as enzymes
that catalyze the hundreds of chemical reactions
necessary for life.
Regulation of protein activity by posttranslational modifications
• After translation, proteins are joined
covalently and noncovalently with other
molecules.
• Complexes that form between lipids and
proteins = lipoproteins,
• Complexes that form between carbohydrates
and proteins = glycoproteins
• Complexes that form between metal ions and
proteins = metalloproteins.
• Post-translational modifications can have both structural and
regulatory functions.
• Important modifications include methylation, acetylation,
ubiquitinylation, and sumoylation.
• The most common regulatory reaction in molecular biology is
the reversible phosphorylation of amino acid side chains.
• Many steps in gene expression and cell signaling pathways
involve posttranslational modification of proteins by
phosphorylation.
• Kinases catalyze the addition of phosphate groups, whereas
enzymes called phosphatases remove phosphates.
• Kinases tend to be very specific, acting on a very few
substrates.
• Phosphatases tend to be nonspecific.
• Many kinases self-regulate through autophosphorylation;
many also can initiate reactions that are part of other
negative feedback systems.
• Two protein kinase groups have been widely studied in
eukaryotes:
– phosphorylate tyrosine side chains,
– phosphorylate serine or threonine side chains.
• Adding phosphate to a protein can cause it to change
its shape, for example by masking or unmasking a
catalytic domain; or the phosphorylated side chain
itself can be part of a binding motif recognized by
other proteins allowing proteins to dock and facilitating
multiprotein complexes to form, or conversely to
promote dissociation of a complex
Molecular chaperones
• Molecular chaperones increase the efficiency of the overall
process of protein folding by reducing the probability of
competing reactions, such as aggregation.
• ATP is required for most of the molecular chaperones to
function with full efficiency.
• Molecular chaperones include heat shock proteins, such as
Hsp40, Hsp70, and Hsp90, which promote protein folding
and aid in the destruction of misfolded proteins
• The designation of these as heat shock proteins reflects the
fact that their concentrations are substantially increased
during cellular stress.
Ubiquitin-mediated protein
degradation
• Cells have several intracellular proteolytic
pathways for degrading:
a) normal proteins whose concentration must
be rapidly decreased,
b) misfolded or denatured proteins,
c) foreign proteins taken up by a cell
Protein misfolding diseases