Protein Synthesis and Structure Flashcards
LO #1: Describe the role of mRNA, tRNA, and ribosomes in protein synthesis and the main
steps of protein translation
The genetic code shows how protein sequence information is stored in nucleic acids. Specific
triplets of nucleotides, termed codons, encode each amino acid in the peptide sequence
that forms a protein’s primary amino acid sequence. In protein translation these nucleotides
are read successively in a non-overlapping fashion. The genetic code is first transcribed into
messengers RNAs (mRNAs) ion the nucleus. The mRNA is then used to create the peptide
sequence through translation. Synthesis of proteins occurs at the surface of the rough
endoplasmic reticulum, so-called because the surface is covered with ribosomes giving it a
rough, rather than a smooth, surface.
Step 1 – Initiation occurs when the protein synthetic machinery selects the appropriate starting
point for reading the mRNA and for forming the peptide bond. The codon for methionine, AUG,
usually is the starting codon so that essentially all proteins begin with a methionine.
Step 2 – Elongation includes the ribosome travelling down the message, reading codons and
bringing in the proper aminoacyl tRNAs to translate the message for the peptide sequence. The
incoming aminoacyl tRNA is brought into the ribosome A site, where it is matched with the
codon that is being presented (Figure 4).
Step 3 – Termination occurs when the ribosome encounters a codon in the A site that signifies
‘stop’. There are slightly different views as to what happens. There may be a release factor
bound to the stop codon, that displaces the ribosome when it reaches that point. Another idea is
that the stop codon has no matching tRNA causing the ribosome to stall. Stalling may then
result in destabilization of the ribosome and release factors then disassemble the ribosome and
cut free the peptide strand.
LO #2: Describe the mechanisms by which antibiotics inhibit protein synthesis
Protein synthesis, a central function in cellular physiology, serves as the primary target of
many antibiotics. Most of these antibiotics inhibit protein synthesis in bacteria with minimal
effect on eukaryotic protein synthesis. Bacterial ribosomes contain a small (30S) and a large
(50S) subunit. These subunits combine to facilitate initiation complex formation. The 50S
subunit alone processes peptide elongation followed by peptide translocation. The 30S subunit
alone introduces a new aminoacyl-tRNA. The basis for the inhibitory action of antibiotics on
protein synthesis requires understanding their mechanisms of action
Aminoglycosides and linezolid, an oxazolidinone, diminish bacterial ribosomal function by
binding to the 30S and 50S subunits, respectively, thereby inhibiting formation of the initiation
complex. Aminoglycosides include streptomycin, gentamicin, neomycin, tobramycin and
amikacin. Chloramphenicol binds to the large ribosomal subunit of bacteria to inhibit the
peptidyl transferase activity and prevent peptide elongation. Clindamycin and macrolids
(e.g., erythromycin) also bind to the 50S subunit but inhibit peptide translocation.
Tetracyclines (e.g., tetracycline, doxycycline, minocycline) bind to the bacterial small
ribosomal subunit to prevent the binding of aminoacyl-tRNAs to the ribosome.
LO #3: Describe the types of post-translational modifications and the nature of the role of each
Proteolytic processing:
Many proteins are initially synthesized as large, inactive precursor polypeptides that are
trimmed by proteases to form their smaller, active forms. Examples include insulin and
collagen molecules, whose processing is described below.
Formation of disulfide cross-links:
After folding into their native conformations, some proteins form intrachain or interchain
disulfide bridges between cysteine residues. This type of modification is common for proteins
that are secreted outside the cells, helping to protect the native conformation from
denaturation in the oxidizing extracellular environment. Therefore, in general, stable disulfide
bonds are formed in the endoplasmic reticulum (ER), the first compartment of the cellular
protein secretory machinery. Insulin processing (below) provides such an example.
Covalent posttranslational modifications STEP 1 RELEVANT
Phosphorylation:
The hydroxyl groups of certain serine, threonine, and tyrosine residues can be reversibly
phosphorylated. The phosphate group adds negative charges to the proteins. The phosphate
is added to proteins by protein kinases and removed by protein phosphatases. The role of
protein phosphorylation varies greatly with the nature and role of each protein, and plays a
key role in cellular signaling cascades.
Glycosylation:
Many secreted proteins undergo covalent attachment of carbohydrate side chains (sugars),
generating glycoproteins. Many proteins that function outside the cells, as well as the
lubricating proteoglycans that coat mucous membranes, contain side chains modified with
sugars. The glycosylation of proteins is catalyzed by glycosyltransferases.
Hydroxylation:
The hydroxylation modification can take place on various amino acids, including proline,
lysine, asparagine, aspartate and histidine, and regulates protein stability. Protein
hydroxylation plays an important role in the maturation of collagen fibers
Methylation:
Generally methylation involves addition of one to three methyl groups to lysine or arginine.
This process, catalyzed by methyltransferases, neutralizes the positive charges of side
groups of these amino acids. Demethylases remove the methyl group. The pattern of
methylation of specific lysine and arginine residues of histones regulates their interaction with
DNA and gene transcription. Histones are proteins that make up nucleosomes around which
DNA wraps around to form chromatin. In other proteins, the carboxyl groups of some
glutamate residues undergo methylation, neutralizing the negative charge of the side group.
Acetylation:
Addition of an acetyl group to an N-terminus or internal lysine residues occurs in a cotranslational fashion and plays an important role in the synthesis, stability and localization of
proteins. Lysine acetylation aids in the regulation of histone proteins and regulation of gene
expression. Acetyltransferases add the acetyl group that is removed by deacetylases.
Lipid modifications:
Lipid moieties of varying nature and length can attach to many proteins. In general, the
covalent linkage of lipids to proteins mediates the anchoring of these proteins to intracellular
membranes. Amino acids that can be modified with lipids include an N-terminal glycine
(myristylation) and cysteine (palmitoylation and prenylation). The linkage formation is
catalyzed by specific lipid transferases.
Prosthetic group:
Many proteins require a covalently bound prosthetic group for their activity. A prosthetic
group is often derived from vitamins in the diet or metal ions. In enzymes, prosthetic groups
often are found in the active site, playing an important role in catalysis. For example the
heme group in myoglobin (Mb) and hemoglobin (Hb) is key to the function of these proteins
and promotes oxygen binding. The role of the heme group in Hb will be discussed further in
upcoming lectures on Hb and Hb disorders.
LO #4: Outline the sequence of events in the synthesis and processing of insulin including
differentiating its precursor forms
Insulin synthesis and packaging of the hormone into secretory granules proceed in an orderly
fashion (Figure 10). Following transcription in the nucleus (step 1), preproinsulin is synthesized
by ribosomes (step 2). The hydrophobic “pre” sequence of preproinsulin is a signal peptide
sequence that ‘signals’ the opening of a channel to allow the peptide through the membrane of
the ER into the lumen (cisternal space) of this organelle. After the channel opens, the signal
sequence guides the growing polypeptide chain into the lumen. Only a polypeptide scheduled
for translocation has access to the channel, thus preventing other substances from arbitrarily
entering the lumen of the ER. Once the signal peptide has entered the lumen, it is removed by a
signal peptidase on the inner surface of the ER membrane. The channel closes after synthesis
of the entire preproinsulin is entirely within the lumen. Chaperones supervise proper folding to
proinsulin. The free SH groups of cysteine residues are oxidized to form disulfide bonds.
After proinsulin forms, transfer vesicles pinch off from the RER to transport proinsulin to the
Golgi (step 3). In the Golgi, proinsulin is packaged into secretory granules with proteases that
remove the amino acids that join C-peptide to the A and B chains of insulin (step 4). Secretory
granules store both insulin and C-peptide (step 5). These granules traverse the cytoplasm
along microtubules toward the plasma membrane (step 6). Upon stimulation of the cell by
glucose, Ca2+ enters the cell. Calcium signals the granules to fuse with the plasma membrane
and induces contraction of microfilaments causing the secretory granules to discharge insulin
and C-peptide into the extracellular fluid by exocytosis (step 7).
LO #5: Describe the regulation of the synthesis and secretion of insulin by glucose
Increased plasma glucose concentration is the most important physiologic regulator of insulin
secretion, which is initiated at glucose greater than 100 mg/dL. Insulin secretion in response to
glucose is biphasic (Figure 11). The first-phase response begins within 1 min, peaks at 3-5 min
and lasts about 10 min. When blood glucose remains high, a second-phase ensues
characterized by a more gradual, prolonged period that terminates soon after removal of the
glucose stimulus. The first-phase causes release of insulin-containing granules at the cell
surface and the second-phase involves secretion of both stored and newly synthesized insulin.
A biochemical model of regulation of insulin secretion depicts the signalling involved in the
process (Figure 12). Glucose enters the β-cell via GLUT-2 (Figure 12, 1), a transporter protein,
and is phosphorylated by glucokinase, which serves as a “glucose sensor” in the β-cell. As a
consequence of glucose metabolism (2), multiple signals are generated. The major result of
glucose metabolism (2) is increased uptake of Ca2+ from the blood into the cytoplasm of the cell.
This portion of the signal is mediated by increased ATP to ADP. The increased ratio causes the
closing of channels that normally facilitate the exit of K+ from the cell (3). Closing of the
channels generates action potentials that open other channels allowing Ca2+ to enter the β-cell.
Glucose also prevents efflux of Ca2+ out of the cell. Additionally glucose increases intracellular
Ca2+ by indirectly causing its release from mitochondria (4). Increase of intracellular Ca2+ causes immediate secretion of insulin (5) by triggering exocytosis of existing secretory granules. Ca2+
also binds to calmodulin (CaM), which activates CaM-dependent kinase to promote insulin
biosynthesis (6). Elevated Ca2+ also increases the activity of protein kinase C (7).
LO #6: Describe the steps and relevant cofactors in the synthesis and processing of collagen
including the post-translational modifications of amino acid residues during its maturation
(i.e., proteolysis, hydroxylation, glycosylation, and oxidation)
Like insulin, collagen is first synthesized as a pre-procollagen form by the ribosomes. The
procollagen chain undergoes processing in the ER and Golgi complex prior to being secreted.
This processing includes hydroxylation, glycosylation and formation of the trimer (Figures 15
and 16). In the ECM, procollagen is converted to collagen by specific proteolytic enzymes that
remove the terminal region of the procollagen chains. The collagen is then assembled to form
the collagen fibril and eventually the collagen fiber, which is formed by bundles of fibrils. Processing of fibrillar collagen occurs via the following steps and post-translational
modifications, which are those changes in the protein that occur after synthesis.
Step 1: Collagen genes are transcribed in the nucleus producing mRNA that is translated by
ribosomes on the RER.
Step 2: As it is synthesized, the pre-procollagen moves into the lumen of the ER where the
signal peptide is cleaved by a signal peptidase to form the procollagen.
Step 3: Proline residues are hydroxylated by prolyl hydroxylase to form hydroxyproline (OHPro). This iron-containing enzyme requires ascorbic acid (vitamin C) as a coenzyme to
maintain the iron in its Fe+2 (ferrous; reduced) state. Hydroxylation of proline causes the
procollagen chain to form the tight helix required to assemble the monomers into a triple helix.
Because prolyl hydroxylase only recognizes single chains, hydroxylation of proline ceases once
the triple helix is formed. Failure to form OH-Pro prevents the monomers from assembling into
mature trimers resulting in accumulation of unprocessed collagen in the ER. Accumulation of monomeric chains eventually leads to cell death. The formation of OH-Pro is tightly coupled to
triple helix formation that inhibits further activity of the enzyme. Hence OH-Pro plays an
important role assembling the individual monomers in the proper register (alignment)
Step 4: Lysine residues are also hydroxylated to hydroxylysine (OH-Lys) by lysyl
hydroxylase that requires, like prolyl hydroxylase, ascorbic acid and iron. Subsequent
glycosylation requires enzymatic attachment of sugars (galactose or galactosyl-glucose) to OHLys. Unlike hydroxylation of proline, the hydroxylation and glycosylation of lysine is not wellregulated (OH-Pro is not glycosylated). The OH-Lys and sugar content of collagens is
determined by how long the procollagen α-chains remain in the ER lumen before they are
assembled into triple helixes. If prolyl hydroxylase is inhibited so that the chains remain longer in
the ER, then the sugar content increases. Increased glycosylation creates a problem, because
the hydroxyl groups on the sugars become hydrated causing collagen to lose its tensile
strength. Potential roles for glycosylation include aiding the secretion to the extracellular matrix
and organization of fibrils. Because fibrillar collagen in babies is processed slowly, it contains
more sugar and has less tensile strength than in adults – hence babies are more ‘flexible’.
Step 5: For efficient triple helix formation, the three procollagen chains need to be properly
aligned. Proper alignment is aided by oxidation of cysteine residues (side chain -CH2-SH) in the
C-terminal non-helical segments to disulfide bridges (-S-S-) that hold the chains in position for
the intertwining of the three chains.
Step 6: After the triple helix forms, procollagen is transported from the ER to the Golgi via
transfer vesicles.
Step 7: The Golgi then packages the procollagen triple helix into secretory vesicles that fuse
with the membrane and secrete their contents to the extracellular matrix for further processing.
Extracellular synthesis and processing:
After secretion, the procollagen is further modified by proteases termed procollagen peptidases
that remove the globular cysteine-rich N- and C- terminal domains, leaving short peptides at
each end. This modification decreases the solubility 1000-fold, causing the collagen units to
spontaneously assemble into fibrils (Figure 17).
Newly formed fibrils are covalently stabilized through crosslinking potentiated by the action of
lysyl oxidase. The primary substrate for lysyl oxidase is hydroxylysine. Oxidation converts the
residues from amines to active aldehydes known as allysines. Lysyl oxidase requires copper
and vitamin B6 as cofactors. Reaction of allysine with free amino groups on other amino acid
residues (e.g, unmodified lysine, hydroxylysine or allysine) eventually forms stable covalent
crosslinks that are vital to the structure of mature collagen fibers. If formation of OH-Lys is
defective then the extent of cross-link formation is markedly diminished and the collagen fibers
fail to mature properly
