From Gene to Protein,

Cellular and sub-cellular protein expression

RNA and protein synthesis
Genetic Code
 Intro to gene expression (central dogma)
Genes specify functional products (such as proteins)
• A DNA molecule is divided up into functional units called genes.
• Each gene provides instructions for a functional product, that is, a molecule needed to perform a job in the cell.
• In many cases, the functional product of a gene is a protein. For example, Mendel's flower color gene provides
instructions for a protein that helps make colored molecules (pigments) in flower petals.

protein-coding genes
How does the DNA sequence of a gene specify a particular protein?

Transcription

RNA polymerase

non-coding strand

central dogma of molecular biology

 RNA polymerase

RNA polymerases are enzymes that transcribe DNA into RNA. Using a DNA template, RNA polymerase builds a new RNA
molecule through base pairing. For instance, if there is a G in the DNA template, RNA polymerase will add a C to the new,
growing RNA strand. RNA polymerase always builds a new RNA strand in the 5’ to 3’ direction. That is, it can only add RNA
nucleotides (A, U, C, or G) to the 3' end of the strand.
RNA polymerases are large enzymes with multiple subunits, even in simple organisms like bacteria. Humans and other
eukaryotes have three different kinds of RNA polymerase: I, II, and III. Each one specializes in transcribing certain classes of
genes. Plants have an additional two kinds of RNA polymerase, IV and V, which are involved in the synthesis of certain small
RNAs.

[What do 5' and 3' mean?]

 Transcription initiation

• To begin transcribing a gene, RNA polymerase binds to the DNA of the gene at a region called the promoter.
• The promoter region comes before (and slightly overlaps with) the transcribed region whose transcription it specifies. It contains
recognition sites for RNA polymerase or its helper proteins to bind to. The DNA opens up in the promoter region so that RNA
polymerase can begin transcription.
• Each gene (or, in bacteria, each group of genes transcribed together) has its own promoter. A promoter contains DNA sequences
that let RNA polymerase or its helper proteins attach to the DNA. Once the transcription bubble has formed, the polymerase can
start transcribing.
 Promoters in bacteria
A typical bacterial promoter contains two important DNA sequences, the -10 and -35 elements.
RNA polymerase recognizes and binds directly to these sequences. The sequences position the polymerase in the
right spot to start transcribing a target gene, and they also make sure it's pointing in the right direction.
Once the RNA polymerase has bound, it can open up the DNA and get to work. DNA opening occurs at the -
10 element, where the strands are easy to separate due to the many As and Ts (which bind to each other using
just two hydrogen bonds, rather than the three hydrogen bonds of Gs and Cs).
 Promoters in humans
In eukaryotes like humans, the main
RNA polymerase in your cells does not
attach directly to promoters like
bacterial RNA polymerase. Instead,
helper proteins
called basal (general) transcription
factors bind to the promoter first,
helping the RNA polymerase in your
cells get a foothold on the DNA.
Many eukaryotic promoters have a
sequence called a TATA box. The TATA
box plays a role much like that of the -
10 element in bacteria. It's recognized
by one of the general transcription
factors, allowing other transcription
factors and eventually RNA polymerase
to bind. It also contains lots of As and
Ts, which make it easy to pull the
strands of DNA apart.
 Elongation
During elongation, RNA polymerase "walks" along one
strand of DNA, known as the template strand, in the 3'
to 5' direction.
For each nucleotide in the template, RNA polymerase
adds a matching (complementary) RNA nucleotide to the
3' end of the RNA strand.
The synthesized RNA only remains bound to the
template strand for a short while, then exits the
polymerase as a dangling string, allowing the
DNA to close back up and form a double helix.
In this example, the sequences of the coding strand,
template strand, and RNA transcript are:
Coding strand: 5' - ATGATCTCGTAA-3'
Template strand: 3'-TACTAGAGCATT-5'
RNA: 5'-AUGAUC...-3' (the dots indicate where
nucleotides are still being added to the RNA strand at its
3' end)
The RNA transcript is nearly identical to the non-
template, or coding, strand of DNA. However, RNA
strands have the base uracil (U) in place of thymine (T),
as well as a slightly different sugar in the nucleotide. So,
as we can see in the diagram above, each T of the
coding strand is replaced with a U in the RNA transcript.
Transcription termination
RNA polymerase will keep transcribing until it gets signals to stop. The process of ending transcription is called termination, and it
happens once the polymerase transcribes a sequence of DNA known as a terminator.

Termination in bacteria
There are two major termination strategies found in bacteria:
Rho-dependent and Rho-independent.

a stable hairpin

C and G nucleotides
 Transcription and RNA processing: Eukaryotes vs. bacteria

splicing
 RNA processing: Eukaryotes

splicing
 Translation
After transcription (and, in eukaryotes, after processing), an mRNA molecule is ready to direct protein synthesis.
The process of using information in an mRNA to build a polypeptide is called translation.

The genetic code

During translation, the nucleotide sequence of an mRNA is translated into the amino acid sequence of a polypeptide.
Specifically, the nucleotides of the mRNA are read in triplets (groups of three) called codons.
There are 61 codons that specify amino acids. One codon is a "start" codon that indicates where to start translation.
The start codon specifies the amino acid methionine, so most polypeptides begin with this amino acid.
Three other “stop” codons signal the end of a polypeptide. These relationships between codons and amino acids are called
the genetic code.
 Steps of translation
Translation: steps
1. Initiation
2. Elongation
3. Termination
 Initiation
•A ribosome (which comes in two
pieces, large and small)
•An mRNA with instructions for the
protein we'll build
•An "initiator" tRNA carrying the first
amino acid in the protein, which is
almost always methionine (Met)
During initiation, these pieces must
come together in just the right way.
Together, they form the initiation
complex, the molecular setup needed
to start making a new protein.
The energy is provided by the cell in
the form of guanosine triphosphate
(GTP), a common "energy currency"
molecule that's similar to the better-
known ATP.
Elongation initiation complex has formed, but before
any amino acids have been linked to make a chain. Our
first, methionine-carrying tRNA starts out in the
middle slot of the ribosome, called the P site. Next to
it, a fresh codon is exposed in another slot, called the A
site. The A site will be the "landing site" for the next
tRNA, one whose anticodon is a perfect
(complementary) match for the exposed codon. The
formation of the peptide bond that connects one
amino acid to another. This step transfers the
methionine from the first tRNA onto the amino acid of
the second tRNA in the A site.
The methionine forms the N-terminus of the
polypeptide, and the other amino acid is the C-
terminus.
Once the peptide bond is formed, the mRNA is pulled
onward through the ribosome by exactly one codon.
This shift allows the first, empty tRNA to drift out via
the E ("exit") site. It also exposes a new codon in the
A site, so the whole cycle can repeat.
a few times up to a mind-boggling 33,000 times! The
protein titin, which is found in your muscles and is the
longest known polypeptide.

[What are the N and C terminus?]

 Termination
Translation ends in a process called termination. Termination happens when a stop codon in the mRNA (UAA,
UAG, or UGA) enters the A site. Stop codons are recognized by proteins called release factors, which fit neatly
into the P site (though they aren't tRNAs). Release factors mess with the enzyme that normally forms peptide
bonds: they make it add a water molecule to the last amino acid of the chain. This reaction separates the chain
from the tRNA, and the newly made protein is released.
Translation "equipment" is very reusable. After the small and large ribosomal subunits separate from the
mRNA and from each other, each element can take part in another round of translation.

Epilogue: Processing
Our polypeptide now has all its amino acids does that mean it's ready to do its job in the cell?
Not necessarily. Polypeptides often need some "edits."
• During and after translation, amino acids may be chemically altered or removed.
• The new polypeptide will also fold into a distinct 3D structure, and may join with other polypeptides to make a
multi-part protein.
• Many proteins are good at folding on their own, but some need helpers ("chaperones") to keep them from
sticking together incorrectly during the complex process of folding.
• Some proteins also contain special amino acid sequences that direct them to certain parts of the cell. These
sequences, often found close to the N- or C-terminus, can be thought of as the protein’s “train ticket” to its
final destination.
 Mutations
Sometimes cells make mistakes in copying
their genetic information,
causing mutations.
Mutations can be irrelevant, or they can
affect the way proteins are made and genes
are expressed.

Substitutions
A substitution changes a single base pair by
replacing one base for another.

There are three kinds of substitution

mutations:
•Silent mutations do not affect the sequence
of amino acids during translation.
•Nonsense mutations result in a stop codon
where an amino acid should be, causing
translation to stop prematurely.
•Missense mutations change the amino acid
specified by a codon.
 Insertions and deletions
An insertion occurs when one or more bases are added to a DNA sequence.
A deletion occurs when one or more bases are removed from a DNA sequence.
inserting or deleting bases may change the "reading frame" of the sequence.
These types of mutations are called frame shift mutations.

methionine (Met), isoleucine (Ile), argenine (Arg), methionine (Met), tyrosine (Tyr), and glycine (Gly).
and asparagine (Asn).
 What happens next?

Epilogue: Processing
Our polypeptide now has all its amino acids does that mean it's ready to do its job in the cell?
Not necessarily. Polypeptides often need some "edits."
• During and after translation, amino acids may be chemically altered or removed.
• The new polypeptide will also fold into a distinct 3D structure, and may join with other
polypeptides to make a multi-part protein.
• Many proteins are good at folding on their own, but some need helpers ("chaperones") to
keep them from sticking together incorrectly during the complex process of folding.
• Some proteins also contain special amino acid sequences that direct them to certain parts of
the cell. These sequences, often found close to the N- or C-terminus, can be thought of as the
protein’s “train ticket” to its final destination.
• Ultimately, it will perform a specific job needed by the cell or organism – perhaps as a
signaling molecule, structural element, or enzyme!
 Protein targeting
Overview of cellular shipping routes
Translation of all proteins in a eukaryotic cell begins in the cytosol (except for a few proteins made in mitochondria and
chloroplasts). As a protein is made, it passes step by step through a shipping "decision tree." At each stage, the protein is checked
for molecular tags to see if it needs to be re-routed to a different pathway or destination.

"address label."

Amino sequence called a

signal peptide
 The endomembrane system and
secretory pathway
Proteins destined for any part of the endomembrane
system (or the outside of the cell) are brought to the ER
during translation
Signal peptides
The signal peptide that sends a protein into the
endoplasmic reticulum during translation is a series of
hydrophobic (“water-fearing”) amino acids, usually found
near the beginning (N-terminus) of the protein. When this
sequence sticks out of the ribosome, it’s recognized by a
protein complex called the signal-recognition particle
(SRP), which takes the ribosome to the ER. There, the
ribosome feeds its amino acid chain into the ER lumen
(interior) as it's made.
In some cases, the signal peptide is snipped off during
translation and the finished protein is released into the
interior of the ER (as shown). In other cases, the signal
peptide or another stretch of hydrophobic amino acids
gets embedded in the ER membrane. This creates a
transmembrane (membrane-crossing) segment that
anchors the protein to the membrane.
Transport through the endomembrane system
In the ER, proteins fold into their correct shapes, and may also
get sugar groups attached to them. Most proteins are then
transported to the Golgi apparatus in membrane vesicles. Some
proteins, however, need to stay in the ER and do their jobs
there. These proteins have amino acid tags that ensure they are
shipped back to the ER if they "escape" into the Golgi.
In the Golgi apparatus, proteins may undergo more
modifications (such as addition of sugar groups) and before
going on to their final destinations. These destinations include
lysosomes, the plasma membrane, and the cell exterior. Some
proteins need to do their jobs in the Golgi (are "Golgi-resident),
and a variety of molecular signals, including amino acid tags
and structural features, are used to keep them there or bring
them back.
If they don't have any specific tags, proteins are sent from the
Golgi to the cell surface, where they’re secreted to the cell
exterior (if they’re free-floating) or delivered to the plasma
membrane (if they’re membrane-embedded). Proteins are
shipped to other destinations if they contain the right molecular
labels. For example, proteins destined for the lysosome have a
molecular tag consisting of a sugar with a phosphate group
attached. In the Golgi apparatus, proteins with this tag are
sorted into vesicles bound for the lysosome.
Targeting to non-endomembrane organelles
Proteins that are made in the cytosol (don't enter ER during translation) may stay permanently in the cytosol.
However, they may also be shipped to other, non-endomembrane destinations in the cell. For instance,
proteins bound for the mitochondria, chloroplasts, peroxisomes, and nucleus are usually made in the cytosol
and delivered after translation is complete.
To be delivered to one of these organelles after translation, a protein must contain a specific amino acid
"address label." The label is recognized by other proteins in the cell, which help transport the protein to the
right destination. As an example, let's consider delivery to the peroxisome, an organelle involved in
detoxification. Proteins needed in the peroxisome have a specific sequence of amino acids called
a peroxisomal targeting signal. The classic signal consists of just three amino acids, serine-lysine-leucine,
found at the very end (C-terminus) of a protein. This pattern of amino acids is recognized by a helper
protein in the cytosol, which brings the protein to the peroxisome.
Mitochondrial, chloroplast, and nuclear targeting are generally similar to peroxisomal targeting. That is, a
certain amino acid sequence sends the protein to its target organelle (or a compartment inside that organelle).
However, the nature of the "address labels" is different in each case.

[Don't mitochondria and chloroplasts have their own ribosomes?]