Gabriel Krotkov
XVI. Protein Synthesis
Biology Notes
3/18/14
A. Basics
1. Without proteins, nothing cellular can function
2. DNA contains the code for protein sequencing.
3. Basic Sequence
a. DNA to mRNA to tRNA to Proteins
b. Transcription & Translation
c. DNA holds info, RNA builds it
4. DNA vs. RNA
a. DNA is doublestranded, while RNA is single stranded
b. Two oxygens in DNA, one in RNA
c. 4 Nitrogen Bases each
i. RNA A,U,C,G
ii. DNA A,T,C,G
5. Differences between organisms
a. In the process below is based on eukaryote
b. Note that prokaryotes, since chromatin is not separated from the cytosol,
can do the process of translation while the RNA is being transcribed.
6. Codons
a. Codons are nucleotide triplets that carry the flow of information from gene
to protein in “triplet code”
b. Note that a series of 3 nucleotides is the smallest the provides coding for
all 20 amino acids: 1 per acid means 4 acids, 2 per acid means 16 acids.
B. Transcription
1. This creates mRNA from DNA
a. The initial RNA produced from any gene is known as a “primary transcript”
2. Occurs in the nucleus
3. Each type of gene is transcribed at different time (only copies what it needs
from DNA)
4. Similar to DNA replication, save in that there is only one strand of RNA
5. Steps
a. DNA unwinds, pried apart by RNA polymerase
i. The DNA sequence where RNA polymerase attaches and initiates the
unwinding is known as the “promoter”
ii. The sequence that signals the end of transcription is known as the
“terminator”; but only in bacteria.
iii. The stretch of DNA to be transcribed is known as a “transcription unit”
iv. Terms “upstream” or “downstream” can be used to describe in what
direction one part of the process is from another: the direction of transcription is
“downstream”
v. Bacteria have only one RNA Polymerase, while Eukaryotes have 3: the
Eukaryotes use the other polymerases to transcribe RNA molecules that aren’t
destined to be proteins.
b. RNA Polymerase attaches to a promoter region, which includes a “start
point”
i. In bacteria, the RNA polymerase itself recognizes and binds to the
promoter region, while in eukaryotes; “transcription factors” (proteins) mediate the
binding. These are known as a transcription initiation complex
c. RNA elongates according to base pair rules
i. Exact opposite – mRNA is complementary, not identical, to its template
ii. Polymerase is adding these nucleotides
iii. See diagram 1 below for chart that describes which letters code for
what
d. RNA pulls away when it reaches the terminator region
i. In the terminator region is a sequence on the DNA that results in what is
called the “polyadenylation signal” in the pre-mRNA (in Eukaryotes: AAUAAA). This
then about 10-35 nucleotides downstream, causes proteins to cut the RNA
transcript free from the polymerase.
ii. In prokaryotes, the polymerase detaches from the DNA immediately
after reading the termination sequence and releases the transcript.
e. DNA winds together
i. This occurs naturally with no proteins to hold it apart: the SSBPs (single
strand binding proteins) depart from the scene.
f. RNA released as a primary transcript (pre-mRNA)
g. Parallel Processing
i. This is performed multiple times simultaneously on a single strand of
DNA on different genes
ii. Also, a single gene can be transcribed into multiple mRNAs by several
molecules of RNA polymerase following each other in order.
6. RNA Processing
a. Overview
i. In eukaryotes, the pre-mRNA is modified after transcription
ii. This is to protect the transcript from digestive proteins in the cytosol,
help ribosomes attach to the 5’ end of the mRNA, and to facilitate transportation
b. Alteration of mRNA ends
i. A 5’ cap, or Methyl G cap, is added to the 5’ end after transcription of the
first 20-40 nucleotides
ii. On the 3’ end, a poly-A tail of 50-250 adenines is added onto the
terminator sequence
c. Splicing
i. Parts of the mRNA are removed – avg. length of transcription is about
27000, but only 1200 nucleotides are required for an avg. size protein of 400 amino
acids.
ii. The noncoding bits of RNA (called introns, for intervening sequences)
are often interspersed between coding portions and are removed for translation.
Presumably this prevents some errors in translation. (Coding regions are called
exons)
iii. The introns are cut out and exons joined together.
iv. Splicing is carried out by spliceosomes, which are massive protein
complexes that contain snRNPs (small nuclear ribonuclearproteins), which
recognize the splice sites. The splicesome interacts with introns to release and
degrade them, and join together the exons. Spliceosomes also have snRNA (small
nuclear RNA) which catalyze these processes.
D. Translation
1. Overview
a. This process turns mRNA into a protein
b. tRNA
i. Each type of tRNA molecule translates a particular mRNA codon into a
particular amino acid.
ii. The tRNA molecule arrives at a ribosome bearing a specific amino acid
at one end, and has an “anticodon” on the other end. The anticodon is a nucleotide
triplet that has complementary base pairs to the codon of the mRNA that it will
attach to. This means the tRNA is identical to the DNA, except with U substituted in.
iii. tRNA is made in the nucleus and then travels to the cytoplasm where it
executes it function, picking up amino acids and delivering them to ribosomes.
iv. tRNA is made of a single RNA strand that is ≈80 nucleotides long (pretty
small), which folds back on itself into a three-dimensional shape (basically an L, or a
J, with the anticodon at the top) via its complementary base pairs. The other end of
the tRNA holds the amino acids (this is the 3’ end)
2. Occurs in ribosomes
a. Ribosomes are constructed of two subunits, which bond during translation
i. One small, one large
ii. Both subunits constructed in nucleolus, with transcribed rRNA and
proteins from the cytoplasm
iii. 2/3rds RNA, 1/3 proteins. Since cells have thousands of ribosomes,
rRNA is the most abundant type of RNA in the cell.
b. Sites
i. There are 3 sites that the ribosome can be divided into
ii. The A site (aminoacyl-tRNA binding site) holds the tRNA carrying the
next amino acid to be bound
iii. The P site (peptidyl-tRNA binding site) holds the tRNA carrying the
polypeptide chain
iv. E site (exit site) is where discharged ribosomes leave the ribosome.
3. Stages
a. Initiation
b. Elongation
c. Termination
4. Initiation
a. tRNA picks up amino acid
i. Aminoacyl-tRNA synthetases (one for each amino acid) matches a tRNA
with its proper amino acid.
ii. Each protein has an active site that fits only one amino acid and tRNA,
and this speeds the two coming together.
iii. Using an ATP, the synthetase covalently binds the two together.
b. Small ribosomal subunit binds to mRNA and tRNA
i. This is accomplished by initation factors, that use GTP to bind tRNA to
the active site
c. Scans for start codon by moving downstream along the mRNA until it
reaches the start codon.
d. tRNA with anistart codon bonds to mRNA
e. The large ribosomal subunit then attaches, completing the translation
initiation complex – this is facilitated by proteins called initiation factors.
5. Elongation
a. mRNA is fed through the ribosome
b. tRNA, brings amino acids to the ribosomes & they bond in the order
specified by the mRNA codons
i. See pickup stage above for more detail.
ii. This is facilitated by the hydrolysis of a GTP, which increases the
accuracy and efficiency of this step.
iii. Many tRNAs are present, but only the one with the appropriate codon is
recognized and accepted.
c. Peptide bonds join each tRNA’d amino acid in sequence
i. rRNA in the large ribosomal subunit catalyzes bond between A site, the
new amino acid and the P site, the polypeptide chain.
d. tRNA detaches from ribosomes to grab more amino acids, and mRNA is
pushed further through the ribosome
i. 5’  3’
e. Sequence ends when stop codon reaches acceptor site
6. Termination
a. When the stop codon reachs the A site, ribosome release the protein in it’s
primary structure, and leave the mRNA.
b. A release factor, which is similar to a tRNA, binds to the stop codon in the A
site, and adds a water molecule instead of an amino acid, which hydrolyzes the
polypeptide.
7. Polyribosomes
a. Once a ribosome is far enough past the start codon, a second ribosome can
attach to the mRNA, and speed the process.
8. Completing the protein
a. Protein folding and post-translational modifications
i. A chaperone protein (literally “chaperonin”) helps the polypeptide fold
correctly.
ii. Occasionally you need to add a molecule for the protein to do its job:
these are post-translational modifications
iii. Also, occasionally polypeptides need to have modifications to their
primary structure: either being cleaved into 2 or more pieces, or removing acids
from the amino end of the chain. Alternately, 2 individual chains can come together.
b. Targeting polypeptides to specific locations
i. Translation always starts in the cytosol, and continues there unless the
polypeptide cues the ribosome to attach itself to the nuclear envelope or ER. (Note
that ribosomes can switch their location, as they are identical)
ii. The polypeptides of proteins destined for the endomembrane system
are marked by a signal peptide, which targets the protein to the ER. This signal
peptide (≈20 amino acids near the leading end) is recognized as it emerges from the
ribosome by a signal-recognition particle (SRP), which escorts the ribosome to the
ER, if needed. Usually, the signal peptide is removed once it has served its purpose
iii. If a ribosome is bound, its produced protein will enter the
endomembrane system rather than be sent free in the cytosol.
iv. Other signal peptides are used to target polypeptides to mitochondria,
chloroplasts, the nucleus, and other organelles. In these cases, however, the
translation is completed before the protein is directed.
E. Mutations
1. Mutation types
a. Point mutation
i. Changes in a single nucleotide pair of a gene
b. Substitutions
i. Type of point mutation. A nucleotide-pair substitution replaces one
nucleotide and its partner with another pair.
ii. Some may be silent, others may turn the protein into missense.
c. Insertions and Deletions
i. A frameshift mutation that throws off everything afterwards by shifting
the frame of reading.
ii. A single nucleotide pair is added or removed, creating a nonsense
mutation.
2. Effect types
a. Missense
i. Message is still readable, but incorrect
ii. Occurs because of a change in primary structure
b. Nonsense
i. Makes no damn sense
c. Silent
i. Change does not alter the protein
ii. Occurs because genetic code changed, but was still translated into the
same amino acid.
3. Mutagens
a. Physical and chemical agents that cause mutations
b. Examples include X-rays, ultraviolet light, etc.
F. Genes
1. Genes occur in all life despite huge variation in expression
2. This leads to a definition of genes as: a region of DNA that can be expressed to
produce a final functional product that is either a polypeptide or an RNA molecule.
G. Regulation of gene expression (beneficial to conserve resources)
1. Bacteria
a. 2 Methods of metabolic control
i. Adjust the activity of enzymes already present. Rather quick, relies on
sensitivity of enzymes to chemical cues that modify their speed.
ii. Adjust the production level of an enzyme: control for the # of enzymes
inside the cell. Controlled by operons
b. Operons
i. When genes are coordinately controlled (a single on-off switch can
control the whole cluster), an “operator” (a segment of DNA) controls the access of
RNA polymerase to the cluster of genes. This entire system: operator, promoter
(which signals for the set of genes), repressor, and gene, constitutes an “operon”
ii. By default, the operon is “on”: polymerase can bind to the promoter and
transcribe the genes. A protein called the “repressor”, however, can halt the operon
by binding to the operator, and preventing RNA Polymerase from binding to the
promoter. Repressors are specific for the operators of each individual operon.
(Allosteric inhibition).
iii. Repressors are created from regulatory genes, which are separate
genes from the operon, and have their own promoter. Corepressors, meanwhile,
operate with a repressor to switch an operon off.
c. Repressible vs. Inducible operons [Negative Gene regulation]
i. Repressible operons have usually active transcription, but can be
inhibited by allosteric regulation. (trp operons)
ii. Inducible operons, however, have inactive transcription, but can be
activation when a small molecule inactivates the active repressor unit of the operon.
d. Positive gene regulation
i. Rather than repressing gene regulation, sometimes a cell will activate the
gene, and induce it to work more.
ii. Under certain conditions, cAMP can accumulate and binds to a
regulatory protein that, once prompted by cAMP, can bind to DNA upstream of the
promoter of the operon it is speeding the production of.
iii. The regulatory protein will then facilitate the binding of RNA
polymerase to the promoter region, and increase the rate of transcription.
iv. When the operon no longer needs to be active, then the [cAMP] will fall,
and the regulatory protein will fall off of the DNA.
2. Eukaryotes
a. Differential Gene Expression
i. A process that allows a cell to specialize, and display only a fraction of its
genes and carry out their specific function
ii. This is particularly important in Eukaryotes, because of the specialized
nature of a eukaryotic organism
iii. Most common control point is transcription, occurring in response to
signals external to the cell.
b. Regulation of Chromatin Structure
i. Chemical modifications to Histones play a direct role in transcription
regulation. The n-terminus of each histone protrudes towards its nucleosome, and
enzymes can add/remove material to these tails. For example, in histone acetylation
(or deacetylation), acetyl is attached (or removed) to lysines in histones. A histone
tail with an acetyl will no longer bind to nucleosomes, promoting the folding of
chromatin into a less compact structure, which is easier to access by proteins.
ii. Other chemical groups can be attached or removed to create various
effects. The presence of a methyl will condense the chromatin, while a phosphate
will loosen it. These discoveries have led to a “histone code hypothesis”, proposing
that specific combinations of modifications, and well as the order in which they
occur, determine the chromatin configuration.
iii. DNA Methylation can also modify the tails of histone proteins, which
marks a gene as “inactive”. This is critical to the cell deciding whether to express a
gene, even through cell division (semiconservative – creates genomic imprinting
wherein a methylated DNA strand will mark its sister for methylation)
iv. Epigenetic inheritance can also make nonpermanent modifications in
the expression of DNA.
b. Regulation of Transcription Initiation
i. Transcription factors are required for eukaryotic RNA polymerase to
begin transcription. These will usually bind together crucial factors, like the TATA
box and the RNA Polymerase, forming the transcription initiation complex. Once this
is constructed, then the polymerase can move along the DNA template.
ii. The rate of gene expression can be modified by the binding of
transcription factors, either activators or repressors, to the control elements of a
group of nucleotides called enhancers.
c. Post-transcriptional regulation
i. RNA processing in the nucleus can modify the message of the mRNA, by
indication which parts are considered introns, and which exons. Regulatory proteins
will bind to regulatory sequences within the primary transcript to indicate this.
ii. Nucleotide sequences in the mRNA can indicate how quickly the cell will
degrade the mRNA in the cytoplasm, and this regulate which proteins are created
and expressed.
iii. During the initiation of translation, regulatory proteins can block mRNA
entering the ribosome by binding to the 5’ or 3’ at either end. Similarly, if a poly-A
tail is not of sufficient length, it cannot attach to the ribosome. Thus, a cell can
“cancel an order” of a protein by eliminating some of the Poly-A tail.
iv. Even after the protein is created, a cell can control some proteins: often
a phosphate is needed to active a protein, and not providing that will simply leave
the protein dormant. Similar modifications to a constructed protein can control gene
expression.
v. Finally, a protein can be marked for quick destruction by attaching
another protein: ubiquitin, to the first protein. Proteasomes, a giant polypeptide,
will then detect and destroy the unneeded protein.
3. Noncoding RNA
a. Only about 1.5% of RNA is coded into proteins; while the rest is used for
various other purposes in the cell. We devote a lot of attention to RNA’s role in
protein synthesis, but that’s far from it. It, among many other things, has a role in
gene regulation.
b. MicroRNA (miRNA) and Small Interfering RNA (SIR)
i. miRNAs are intended to bind to complementary sequences in mRNA
molecules. They are made from a longer RNA precursor that folds back on itself,
forming doubled-stranded “hairpin” structures that are broken up by enzymes to
form a double-stranded 22 nucleotide pair. One of the 2 strands degrades, and the
other is now miRNA.
ii. miRNA can made a complex with one or more proteins, allowing the
complex to bind to any mRNA that has a small series of a complementary sequence,
degrading or blocking the translation of the substrate.
iii. siRNAs are similar in size and function, but form in another way: SIRs
form from a long, linear, double-stranded, RNA; while the miRNAs are formed from
hairpin structures.