Download Proteins – synthesis and roles in cells

Proteins and Nucleic Acids
Also featuring amino acids and
polypeptides…..
Nucleic Acids
• DNA and RNA are
informational molecules in
eukaryotic cells.
• They are built of
nucleotides (the purines
guanine and adenine and
the pyrimidines cytosine,
thymine and uracil) with a
sugar-phosphate
backbone.
• DNA has C-G and A-T;
RNA has C-G and A-U.
Nucleic Acids
Polymerization to
form the nucleic acid
chain
(As with other
polymers, the linkage
reaction involves
hydrolysis. In this case
a phosphodiester
bond is formed.)
Crosslinks in the complementary pairing
between purines and pyrimidines
Hydrogen bonds stabilize the pairs of nucleotides in the
DNA double helix.
Transcription: DNA ->RNA
• 1. Binding: RNA polymerase binding to a DNA
promoter sequence triggers localized unwinding
of the double helix.
• 2. RNA polymerase initiates synthesis of
messenger RNA from one of the two DNA
strands.
• 3. Elongation of the RNA from complementary
nucleotides occurs as the RNA polymerase
moves along the DNA.
• 4. RNA polymerase reads a termination
sequence and causes the completed mRNA
strand to dissociate from its DNA template.
DNA transcription
Polytene chromosomes reveal
DNA regions that are undergoing
transcription in a dipteran insect.
Highly condensed DNA,
known as heterochromatin:
Arrows indicate the
condensed X chromosome
(Barr body) at the periphery
of the nucleus of a female
mammal.
Posttranscriptional Processing
• After a primary transcript mRNA has been
completed,
• Introns – segments of the primary
transcript that will not contribute to the
protein sequence – are clipped out.
• The remaining exons are spliced together
to form the mature mRNA.
Posttranscriptional processing of the mRNA for the
beta chain of hemoglobin
Splice variants
• In many cases, the exons can be cut and
spliced in a number of different ways –
leading to splice variants. Splice variation
is one way in which a single gene can give
rise to multiple distinct proteins. Gene
splicing is observed in high proportion of
genes. In human cells, about 40-60% of
the genes are known to exhibit alternative
splicing.
Mechanisms that cause splice variants
• There are several types of common gene splicing events.
• Exon Skipping: This is the most common known gene splicing
mechanism in which exon(s) are included or excluded from the final
gene transcript leading to extended or shortened mRNA variants.
The exons are the coding regions of a gene and are responsible for
producing proteins that are utilized in various cell types for a number
of functions.
• Intron Retention: An event in which all or part of an intron is
retained in the final transcript. In humans 2-5 % of the genes have
been reported to retain complete introns and about 95% retain small
parts of introns.
• Alternative 3' splice site and 5' splice site: Alternative gene
splicing includes joining of different 5' and 3' splice site. In this kind
of gene splicing, two or more alternative 5' splice site compete for
joining to two or more alternate 3' splice site.
• The consequences of alternative splicing may be trivial, functional or
pathological.
It’s time to shift
from nucleic
acids to amino
acids, peptides
and proteins…
The relationship between nucleic acids and
proteins was forged over a period of time in the
dim past, and recent appreciation that some
mitochondria, protoza and the Archaea differ from
multicellular animal cells in details of the triplet
code amino acid specification attest to splits in the
ancestry during the period of “experimentation”.
Why only 20 amino acids?
• There are at least 70 different amino acids, most of
which are not found in proteins.
• At some very early state of evolution of life, a
commitment to use only L amino acids was made. This
cuts the number of possibilities by half.
• Some of the “forbidden” L amino acids are toxic.
The nature of amino acids
•
The building blocks of peptides and proteins can be
divided into four categories, based on how they will
interact within the protein structure/aqueous medium.
1.
Nonpolar (10) : do not interact with water- located in
interior of soluble proteins, or on exterior of
intramembrane domains of membrane proteins
Polar (5) : hydrophilic, located in protein exterior for
soluble proteins, or in interior of intramembrane
domains of membrane proteins
Basic (3) : hydrophilic, form hydrogen bonds with water
Acidic (2) : very hydrophilic, usually located on the
protein surface
2.
3.
4.
Amino
acids
A different text
gives a different
breakdown: 9
nonpolar, 6
polar, etc. It is
cysteine that
seems to not
know where to
go... .
Amino Acid Functions
• Singaling molecules e.g. glycine, GABA (a
glutamine derivative) and dopamine (a
tyrosine derivative) are neurotransmitters
• Metabolizable for energy
• Sources of amine group in synthesis
• Peptide and protein subunits
Peptides
Small chains of amino acids (fewer than 40
amino acids – the smallest is 3 amino acids)
serve as peptide hormones and
neurotransmitters. Examples :
•
Oxytocin 9 amino acids CYIQNCPLG (C's are disulfide
bonded). Uterine contraction, causes milk ejection in
lactating females, responds to suckling reflex and
estradiol, lowers steroid synthesis in testes
• Vasopressin antidiuretic hormone, ADH) 9 amino acids
CYFQNCPRG (C's are disulfide bonded) Responds to
osmoreceptor which senses extracellular [Na+], blood
pressure regulation, increases H2O readsorption from
distal tubules in kidney
• Melanocyte-stimulating hormones (MSH): a peptide = 13
amino acids, b polypeptide = 18 amino acids, g
polypeptide = 12 amino acids. Pigmentation
Polypeptides are longer strings of amino acids (typically
between 100 and 1000 peptide residues)
• Amino acids are joined by peptide bonds between the alpha amino
acid group (N terminus group) of one amino acid and the alpha
carboxyl group (C terminus group) of the next amino acid.
• Peptide bonds are formed in a dehydration reaction.
• The sequence of amino acids is listed from the amino to the
carboxyl end:
Translation: Three steps in protein synthesis
1. messenger RNAs
(mRNAs) code for
the amino acids of a
polypeptide based
on a triplet code.
There are 3 stop codons and
one initiation codon.
(Note that there is not a 1:1
match of triplets and amino
acids – there are 64 triplets
and only 20 amino acids, so
the code is redundant –
some amino acids are coded
for by multiple triplets.)
Translation, con’t.
2. Transfer RNAs (tRNAs)
recognize amino acids
(actually, a specific
enzyme is required to
make the attachment)
and bring them to
ribosomes, where they
line them up based on
their complementarity
to the mRNA: GUG
with CAC, etc.
Translation, con’t.
• 3. mRNA is translated into polypeptides on ribosomes,
which are composed of ribosomal RNA (rRNA) and
ribosomal proteins.
Protein Structure: Four levels of organization
Chaperones: Protein folding is generally not
spontaneous
• While the sequence of nucleotides in DNA is
being translated into a sequence of amino acids
to form a protein, the charged groups in the
chain of amino acids are interacting, folding to
allow + to meet –, or hydrophobic groups to cling
together to avoid water. Some proteins assume
their tertiary shape spontaneously, but in others,
this process is assisted by chaperones, proteins
that temporarily stabilize the incomplete protein
by blocking associations that would interfere with
the bending pattern characteristic of the
functional protein.
Diseases related to protein misfolding, aggregation
and precipitation
• Alzheimer’s Disease: Plaques of
β amyloid result from
aggregation and precipitation of
partially folded 40-residue
protein fragments; the presence
of a misfolded fragment initiates
aggregation of similar
fragments.
• Bovine spongioform
encephalopathy (mad cow
disease) and the related scrapie
in sheep and Creutzfeldt-Jakob
in humans are caused by prions.
The prion protein normally plays
a function in synapse
modification in learning, but the
abnormally folded proteins form
insoluble fibrous aggregates.
Prion protein
Normal
Diseased
Protein misfolding occurs frequently in
normal cells
• Chaperones don’t always prevent
misfolding
• Misfolded proteins are labeled for
destruction by mechanisms that are
involved in turnover of appropriately folded
proteins (i.e. proteasomes and lysosomes,
which you will hear about in a subsequent
lecture)
Protein Functions
• Enzymes – Catalysts
• Regulation – transcription factors, protein
hormones
• Transport – hemoglobin for O2, membrane
transport proteins
• Storage – Ferritin can carry 4,500 iron
molecules
• Contraction – actin, myosin, tubulin
• Structural – keratin, collagen
• Protective – blood clotting, IGG’s
Posttranslational Processing
• In posttranslational processing, parts of the
protein structure are removed. It may be as
simple as removal of the signal sequence that
directs a protein to be secreted, or as complex
as what happens to proopiomelanocortin
(POMC) to yield adrenocorticotrophic hormone
(ACTH) (shown in the next slide).
• Like posttranscriptional processing,
posttranslational processing enables one gene
to have multiple products, violating the one gene
– one protein rule that used to be a central
dogma of molecular biology.
Post-translational processing of
proopiomelanocortin (POMC).
POMC in mammals consists of 3
exons, of which exons 2 and 3 are
translated. Prohormone convertases 1
and 2 (PC1/2) break the parent POMC
peptide into successively smaller
peptides by cleavage at paired dibasic
amino acid residues consisting of
lysine (K) and/or arginine (R). The final
products are generated in a tissue
specific manner, for example α-MSH
and ACTH are not produced by the
same cells in the pituitary. The final
products include the melanocortins
(MSHs and ACTH), β-endorphin (βend) and corticotrophin-like
intermediate peptide (CLIP). There are
intermediate peptides whose biological
function remains unclear, such as β
and γ lipotrophins (β-LPH, γ-LPH).
Millington, Nutrition & Metabolism
2007, 4:18.
POMC is a single gene product that
regulates many body activities
• Depending on the type of neuron that is
expressing it, POMC is converted to
different endproducts that regulate a wide
variety of activities, including feeding,
stress responses, metabolism, pain
perception, body pigmentation, sexual
behavior, lactation, etc.
Insulin is another example of a protein that
is created by postranslational processing
In this process, the immature protein is secreted into the endoplasmic reticulum
as it is synthesized. Within the ER, the signal sequence is then removed. The
segments of the original gene product that will become the alpha and beta
chains of the final hormone are folded by a chaperone protein and inked by
disulfide bonds to form proinsulin. Removal of the C-peptide loop connecting the
two chains results in the final hormone.
Summary
Nucleic acids store information (DNA) and
transfer this information into proteins (mRNA, tRNA,
rRNA)
Proteins are the most diverse class of molecules in cells;
their roles are to execute the information carried in the
DNA
Protein function depends on protein structure. The 1o
structure of a protein is established by
posttranscriptional and posttranslational processing,
and provides the basis for higher structural orders,
which generally cannot be attained without the
assistance of other proteins.