Download DNA to Protein

Chapter 7
From DNA to Protein
DNA to Protein
DNA acts as a “manager” in the
process of making proteins
 DNA is the template or starting
sequence that is copied into RNA
that is then used to make the protein

Central Dogma

One gene – one protein
Central Dogma
This is the same for bacteria to humans
 DNA is the genetic instruction or gene
 DNA  RNA is called Transcription



RNA chain is called a transcript
RNA  Protein is called Translation
Expression of
Genes


Some genes are
transcribed in
large quantities
because we need
large amount of
this protein
Some genes are
transcribed in
small quantities
because we need
only a small
amount of this
protein
Transcription
Copy the gene of interest into RNA
which is made up of nucleotides linked
by phosphodiester bonds – like DNA
 RNA differs from DNA

Ribose is the sugar rather than
deoxyribose – ribonucleotides
 U instead of T; A, G and C the same
 Single stranded

 Can
fold into a variety of shapes that allows
RNA to have structural and catalytic functions
RNA Differences
RNA Differences
Transcription

Similarities to DNA replication
Open and unwind a portion of the DNA
 1 strand of the DNA acts as a template
 Complementary base-pairing with DNA


Differences

RNA strand does not stay paired with DNA


DNA re-coils and RNA is single stranded
RNA is shorter than DNA
 RNA
is several 1000 bp or shorter whereas
DNA is 250 million bp long
Template to Transcripts

The RNA transcript is identical to the NONtemplate strand with the exception of the T’s
becoming U’s



Catalyzes the formation
of the phosphodiester
bonds between the
nucleotides (sugar to
phosphate)
Uncoils the DNA, adds
the nucleotide one at a
time in the 5’ to 3’
fashion
Uses the energy
trapped in the
nucleotides themselves
to form the new bonds
RNA
Polymerase
RNA Elongation



Reads template 3’
to 5’
Adds nucleotides
5’ to 3’ (5’
phosphate to 3’
hydroxyl)
Synthesis is the
same as the
leading strand of
DNA
RNA Polymerase

RNA is released so we can make many
copies of the gene, usually before the first
one is done

Can have multiple RNA polymerase molecules on
a gene at a time
Differences in
DNA and RNA Polymerases




RNA polymerase adds ribonucleotides not
deoxynucleotides
RNA polymerase does not have the ability to
proofread what they transcribe
RNA polymerase can work without a primer
RNA will have an error 1 in every 10,000
nucleotides (DNA is 1 in 10,000,000
nucleotides)
Types of RNA
messenger RNA (mRNA) – codes for
proteins
 ribosomal RNA (rRNA) – forms the core
of the ribosomes, machinery for making
proteins
 transfer RNA (tRNA) – carries the amino
acid for the growing protein chain

DNA Transcription in Bacteria
RNA polymerase must know where the
start of a gene is in order to copy it
 RNA polymerase has weak interactions
with the DNA unless it encounters a
promoter


A promoter is a specific sequence of
nucleotides that indicate the start site for RNA
synthesis
RNA Synthesis



RNA pol opens
the DNA double
helix and creates
the template
RNA pol moves nt
by nt, unwinds the
DNA as it goes
Will stop when it
encounters a
STOP codon,
RNA pol leaves,
releasing the RNA
strand
Sigma () Factor
Part of the bacterial RNA polymerase that
helps it recognize the promoter
 Released after about 10 nucleotides of
RNA are linked together
 Rejoins with a released RNA polymerase
to look for a new promoter

Start and Stop Sequences
DNA Transcribed



The strand of DNA transcribed is dependent on which
strand the promoter is on
Once RNA polymerase is bound to promoter, no option but
to transcribe the appropriate DNA strand
Genes may be adjacent to one another or on opposite
strands
Eukaryotic Transcription



Transcription occurs in the nucleus in eukaryotes,
nucleoid in bacteria
Translation occurs on ribosomes in the cytoplasm
mRNA is transported out of nucleus through the
nuclear pores
RNA Processing
Eukaryotic cells process the RNA in the
nucleus before it is moved to the
cytoplasm for protein synthesis
 The RNA that is the direct copy of the
DNA is the primary transcript
 2 methods used to process primary
transcripts to increase the stability of
mRNA being exported to the cytoplasm

RNA capping
 Polyadenylation

RNA Processing


RNA capping happens at the 5’ end of the RNA,
usually adds a methylgaunosine shortly after RNA
polymerase makes the 5’ end of the primary
transcript
Polyadenylation modifies the 3’ end of the primary
transcript by the addition of a string of A’s
Coding and Non-coding Sequences



In bacteria, the RNA made is translated to a protein
In eukaryotic cells, the primary transcript is made of
coding sequences called exons and non-coding
sequences called introns
It is the exons that make up the mRNA that gets
translated to a protein
RNA Splicing



Responsible for the removal of the introns to create the
mRNA
Introns contain sequences that act as cues for their
removal
Carried out by small nuclear riboprotein particles
(snRNPs)
snRNPs


snRNPs come
together and cut out
the intron and rejoin
the ends of the RNA
Intron is removed as
a lariat – loop of
RNA like a cowboy
rope
Benefits of Splicing

Allows for genetic recombination


Link exons from different genes together to create a
new mRNA
Also allows for 1 primary transcript to encode for
multiple proteins by rearrangement of the exons
Summary
RNA to Protein
Translation is the process of
turning mRNA into protein
 Translate from one “language”
(mRNA nucleotides) to a second
“language” (amino acids)
 Genetic code – nucleotide
sequence that is translated to
amino acids of the protein

Degenerate DNA Code


Nucleotides read 3 at a time meaning that
there are 64 combinations for a codon (set of
3 nucleotides)
Only 20 amino acids

More than 1 codon per AA – degenerate code with
the exception of Met and Trp (least abundant AAs
in proteins)
Reading
Frames

Translation can occur in 1 of 3 possible reading
frames, dependent on where decoding starts in
the mRNA
Transfer RNA
Molecules



Translation requires an
adaptor molecule that
recognizes the codon on
mRNA and at a distant
site carries the
appropriate amino acid
Intra-strand base pairing
allows for this
characteristic shape
Anticodon is opposite
from where the amino
acid is attached
Wobble Base
Pairing


Due to degenerate code for amino acids some
tRNA can recognize several codons because
the 3rd spot can wobble or be mismatched
Allows for there only being 31 tRNA for the 61
codons
Attachment of AA to tRNA
Aminoacyl-tRNA synthase is the
enzyme responsible for linking the
amino acid to the tRNA
 A specific enzyme for each amino acid
and not for the tRNA

2 ‘Adaptors’ Translate
Genetic Code to Protein
2
1
Ribosomes


Complex machinery that
controls protein synthesis
2 subunits

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
1 large – catalyzes the peptide
bond formation
1 small – binds mRNA and
tRNA
Contains protein and RNA

rRNA central to the catalytic
activity


Folded structure is highly
conserved
Protein has less homology and
may not be as important
Ribosome Structures


May be free in cytoplasm or attached to the ER
Subunits made in the nucleus in the nucleolus and
transported to the cytoplasm
Ribosomal Subunits



1 large subunit – catalyzes the formation of the peptide bond
1 small subunit – matches the tRNA to the mRNA
Moves along the mRNA adding amino acids to growing
protein chain
Ribosomal Movement
E-site

4 binding sites


mRNA binding site
Peptidyl-tRNA binding site (P-site)


Aminoacyl-tRNA binding site (A-site)


Holds tRNA attached to growing end of the peptide
Holds the incoming AA
Exit site (E-site)
3 Step Elongation
Phase
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Elongation is a cycle of events
Step 1 – aminoacyl-tRNA comes into
empty A-site next to the occupied P-site;
pairs with the codon
Step 2 – C’ end of peptide chain
uncouples from tRNA in P-site and links
to AA in A-site

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Peptidyl transferase responsible for bond
formation
Each AA added carries the energy for the
addition of the next AA
Step 3 – peptidyl-tRNA moves to the Psite; requires hydrolysis of GTP

tRNA released back to the cytoplasmic pool
Initiation Process
Determines whether mRNA is
synthesized and sets the reading frame
that is used to make the protein
 Initiation process brings the ribosomal
subunits together at the site where the
peptide should begin
 Initiator tRNA brings in Met


Initiator tRNA is different than the tRNA that
adds other Met
Ribosomal Assembly
Initiation Phase

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Initiation factors (IFs) catalyze the steps
– not well defined
Step 1 – small ribosomal subunit with
the IF finds the start codon –AUG



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Moves 5’ to 3’ on mRNA
Initiator tRNA brings in the 1st AA which is
always Met and then can bind the mRNA
Step 2 – IF leaves and then large
subunit can bind – protein synthesis
continues
Met is at the start of every protein until
post-translational modification takes
place
Eukaryotic vs Procaryotic

Procaryotic



No CAP; have specific ribosome binding site upstream of AUG
Polycistronic – multiple proteins from same mRNA
Eucaryotic

Monocistronic – one polypeptide per mRNA
Protein Release

Protein released when a STOP
codon is encountered

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
UAG, UAA, UGA (must know these
sequences!)
Cytoplasmic release factors bind
to the stop codon that gets to the
A-site; alters the peptidyl
transferase and adds H2O
instead of an AA
Protein released and the
ribosome breaks into the 2
subunits to move on to another
mRNA


As the ribosome
moves down the
mRNA, it allows for
the addition of
another ribosome
and the start of
another protein
Each mRNA has
multiple ribosomes
attached,
polyribosome or
polysome
Polyribosomes
Regulation of Protein Synthesis

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Lifespan of proteins vary, need
method to remove old or
damaged proteins
Enzymes that degrade proteins
are called proteases – process
is called proteolysis
In the cytosol there are large
complexes of proteolytic
enzymes that remove damaged
proteins
Ubiquitin, small protein, is added
as a tag for disposal of protein
Protein Synthesis
Protein synthesis takes the most energy
input of all the biosynthetic pathways
 4 high-energy bonds required for each
AA addition

2 in charging the tRNA (adding AA)
 2 in ribosomal activities (step 1 and step 3
of elongation phase)

Summary
Ribozyme


A RNA molecule can fold
due to its single stranded
nature and in folding can
cause the cleavage of other
RNA molecules
A RNA molecule that
functions like an enzyme
hence ribozyme name