Genetic Code

1
Genetic Code
DNA mRNA
protein
transcription
translation
genetic code means for converting DNA sequence
into protein sequence the original question has
always been how to convert 4 nucleotide bases
into 20 types of amino acids in the 1940's
Beadle and Tatum begain studying a bread mold
Neurospora and isolated mutants (ie. strains
of yeast with damaged genes) that could not
grow when provided with minimal nutrients but
survived OK when complete, or rich nutrients
were provided. Beadle and Tatum identified many
mutants for various products-- amino acids,
vitamins, etc.
2
Genetic Code
They already knew that there were multiple steps
in synthesizing a particular product ie.
perhaps 5 genes for synthesizing isoleucine By
substituting different intermediates into minimal
growth conditions, they could infer which
steps (enzymes) were defective
A B C
D E
W X Y
Z
Beadle and Tatum isolated various strains that
required E for growth By adding A, B, C, or D to
minimal media they could guess which step was
defective. for example, if one strain would grow
when C, D, or E were added to minimal media
but NOT A or B, that means that enzyme Y can
convert C to D and enzyme Z can convert D to
E. However, B cannot be made into C, saying
that the defect was in enzyme X.
3
Genetic Code
One gene, one enzyme hypothesis each gene that
they mutated coded for exactly one single
enzyme so now there is a connection between
mutations and enzyme function We now know this
is slightly off-- one gene codes for 1 protein
some enzymes have 2 or more proteins in them (ie.
F-type ATPases, etc) We also know it goes even
further-- one gene codes for 1 protein and they
do not have to have an enzymatic function-- ie.
actin, hemoglobin, etc. None of these
experiments, though, adressed the question of HOW
a gene codes for a protein (note that at this
point in 1940's, DNA was just determined to be
the genetic material)
4
Genetic Code
4 nucleotides in DNA have to somehow code for 20
amino acids 1 nucleotide clearly not
sufficient-- that gives on 4 amino acids 2
nucleotides is better, but not enough-- 42 gives
16 amino acids 3 nucleotides is the minimum-- 43
gives 64 possible amino acids, enough early
1960's, Crick, Brenner and students used acridine
dyes to generate mutants defective for various
enzymes acridine dyes are a mutagen (chemical
that causes mutations) that cause addition or
deletions of single base pairs of DNA additional
acridine dye treatments could sometimes return
enzyme function-- changes were additive--
multiple changes gave active enzyme frameshift
mutation change in DNA sequence which alters the
nucleotide 'letters' making up the amino acid
'words' of a protein
5
Genetic Code
Crick and Brenner showed that '' mutants were
cancelled by '-' mutants Two '' or two '-'
mutants did not cancel Three '' or three '-'
mutants WERE able to cancel out each other, just
like a '' and a '-' this suggested a
'triplet' code-- 3 nucleic acids per amino acid
'' frameshift and '-' frameshift nearby gives
mostly normal enzymes two '' or two '-'
enzymes could not give a readable message
three '' or '-' mutations near each other would
add () or remove (-) one amino acid,
change a few others, and leave the rest of the
protein Crick and Brenner saw these reversions
(returns to normal) frequently and they knew
there were 64 possible 3 nucleotide codes to make
20 amino acids
6
Genetic Code
AUG GTC AAT AAA CCG... met val asn lys pro AUG
TGT CAA TAA ACC G... met cys gln OCR AUG TTG TCA
ATA AAC CG... met phe ser ile asn AUG TTT GTC
AAT AAA CCG... met phe val asn lys pro
normal protein sequence
one mutation
two () mutation
three () mutation note the sequence similarity
7
Genetic Code
degenerate code one amino acid can be coded for
by more than one triplet code ie synonyms
two 'words' meaning same thing Note that these
arguments mean that the code is
non-overlapping an overlapping code would have
nucleotides 1-3 coding for the first amino
acid, nucleotides 2-4 coding for the second amino
acid, etc. in an overlapping code, the '' or
'-' mutants could only change a few amino
acids-- all the others would be unaffected there
are a few cases (usually viruses) that have
overlapping genes ie. genes that share
different reading frames using the same
nucleotides almost always use opposite strands
of DNA
8
Genetic Code
Nirenberg and Matthei developed biochemical
system outside of cells to study protein
synthesis in their system, if they added RNA
they would see more protein made used an enzyme
called polynucleotide phosphorylase to make RNA
sequence composed of only 1 type of base, either
G, C, A, or U (not T!) UTP
poly(U), ATP poly(A)
etc with poly(U) added to their cell free
system, they saw more phenylalanine
incorporated into proteins Reasoned that UUU
coded for phenylalanine showed AAA coded for
lysine, CCC for proline, and GGG for glycine
pnp
pnp
9
Genetic Code
Note that this brings up an issue-- DNA is double
stranded ie. GACGTCTAG CTGCAGATC one
strand will serve as the template-- strand that
is used to direct the synthesis of the
RNA ie. if GACGTCTAG is the template DNA, it
would direct the synthesis of CUGCAGAUC using
the complimentary base pairs AU and GC, the
same rules as with base pairings within
DNA coding strand DNA strand that is most
similar to the synthesized RNA
10
Genetic Code
codon 3 letter mRNA triplet 'read' by the
protein synthesis machinery bases are always
read starting at the 5' phosphate toward the 3'
end (same order that nucleotide chains are
made in) 5'-AUGUUUCGCAGA-3' mRNA (like the
coding strand) 3'-TACAAAGCGTCT-5' DNA template
strand H. Gobind Khorana, instead of using
polynucleotide phosphorylase, synthesized
RNAs with precise sequences arranging various
possible orders together, they could identify all
codons
11
Genetic Code
12
Genetic Code
several special codons in the genetic code AUG
(in DNA represented by ATG) coding for
methionine-- initiator codon starts the
process of protein synthesis 3 termination
codons (UAA, UAG, UGA) or stop codons 3 base
code to end protein synthesis the genetic
code is unambiguous-- a 3 base codon always make
the same amino acid wobble base the third
base pair in a codon can often be changed
without changing the protein sequence Almost all
organisms use the exact same code a few
exceptions that make a non-standard amino acid or
use a special transfer RNA (tRNA) that
changes the meaning of a codon
13
Transcription in Prokaryotes
RNA polymerase enzyme which synthesizes mRNA
from the DNA template strand using G, C, A,
and U (uracil) as the bases core enzyme of RNA
polymerase is a tetramer with 2 a and 2 b
subunits holoenzyme core RNA polymerase plus
the sigma factor s sigma factor recognizes
sequences of DNA that precede coding
DNA promoter regulatory sequence of DNA before
the coding region of a gene extremely
important for regulating what genes are turned
on relatively simple in prokaryotes (discussed
more in Chapter 23) different sigma factors
recognize different promoters allows bacteria
to turn on particular genes only when they're
needed!
14
RNA polymerase (T7 Virus)
single stranded DNA
double stranded DNA
15
RNA polymerase (T7 Virus)
Note the nice little hole for the single stranded
DNA to slide through
16
Transcription in Prokaryotes
4 steps of transcription Binding, Initiation,
Elongation, and Termination transcription unit
segment of DNA that gives rise to a RNA
molecule 1) RNA core enzyme (recognizing the s
factor bound to a promoter) binds to the DNA
at that site binding initiates unwinding of
the DNA double helix upstream DNA 5' of the
start of RNA transcription (ie. does NOT get
included in the RNA chain-- usually contains the
promoter region) downstream DNA 3' of start of
RNA transcription (included in the RNA) promoter
binding unwinds 15-18 bp of the DNA near where
transcription begins
17
DNA footprinting
promoters were originally identified by DNA
footprinting DNA footprinting general technique
for identifying sites on the DNA that are
bound by proteins if a protein is bound to the
DNA, a chemical or enzyme that breaks
phosphodiester bonds cannot reach the portion of
DNA bound to the protein-- that region is
protected you then randomly fragment the DNA
region (having isolated it earlier along with
the protein of interest) and separate the pieces
by electrophoresis (remember-- phosphate is
negatively charged so will move in an electric
field!) regions of DNA with less fragmentation
have proteins bound to them
18
(No Transcript)
19
DNA footprinting
20
Transcription in Prokaryotes
2) After DNA binds the s factor, RNA polymerase
initiates transcription recognizes s factor
the unwound DNA NTPs (ie. ATP, CTP, GTP, or
UTP) hydrogen bond to the template strand of
the DNA in the first 2 positions RNA polymerase
catalyzes formation of a phosphodiester bond
between first 2 nucleotides, joining the 3'
hydroxyl of the first base to the 5' phosphate
of the second base
generates a phosphodiester bond and inorganic
phosphate (PP)
21
Transcription in Prokaryotes
RNA polymerase always starts at the 5' end and
moves to the 3' ie. new bases are added to the
free 3' hydroxyl group of ribose PP is lost
from the newly added NTP polymerase moves along,
forming phosphodiester bonds as NTPs bind after
about 9 bp, s factor detaches from the RNA
polymerase-- initiation is complete 3)
Elongation RNA polymerase moves happily along
the DNA moves 5' to 3' -- NTPs bind to the 3'
OH, giving off PP DNA is unwound as the
polymerase moves forward winds back up after
it passes-- RNA doesn't form double helices as
well (about 12 bp only) RNA strand grows and
exists on its own
22
Transcription in Prokaryotes
4) Termination RNA polymerase stops adding
bases termination signal sequence of DNA that
makes RNA polymerase halt 2 types of termination
signals GC rich followed by several U's GC
rich region is complimentary to itself-- forms a
hairpin hairpin nucleic acid structure that can
base pair to itself
23
Transcription in Prokaryotes
rho (r) factor protein that binds to a specific
50-90 bp sequence of RNA rho binding unwinds RNA
from the DNA template, essentially pulling it
away from the DNA and causes the RNA and
polymerase to 'fall off' the DNA once the RNA
polymerase core enzyme falls off DNA, can bind to
a new sigma factor and start the process again
(and again, and again!) at the same or
different promoters note that RNA polymerase is
an ENZYME-- it isn't changed by making the
phosphodiester bonds
24
Transcription in Eukaryotes
follows the same 4 stages binding, initiation,
elongation, termination because the organisms
are more complex, so is transcription instead of
1 RNA polymerase, there are now 3, each with
different characteristics RNA polymerase I
(RNApol I) makes ribosomal RNAs (rRNA) RNApol
II synthesizes messenger RNA for protein
coding also makes small nuclear RNAs for mRNA
processing synthesizes broadest variety of
RNAs RNApol III makes transfer RNA (tRNA) and
other short RNAs all 3 are large multisubunit
enzymes (8-10 subunits) homologous to
the prokaryotic ones
25
Transcription in Eukaryotes
3 different classes of polymerase-- therefore 3
classes of promoters RNA pol I promoter has 2
parts core promoter minimal set of DNA bases
to start rRNA synthesis works, but is not
very efficient upstream control element or
upstream enhancer, is upstream of the core
promoter, binds different proteins, and increases
transcription
RNA pol I
DNA
rRNA
26
Transcription in Eukaryotes
RNA pol II promoter is the most complicated
(because of the diversity of RNA it needs to
make) 1) short Initiator region (Inr) at the
transcription start point 2) TATA box (A-T rich
region) about 25 bp upstream from the Inr 3)
TFIIB recognition element (BRE) immediately
upstream from TATA 4) downstream promoter
element (DPE) 30 bp downstream from Inr Not
every promoter has to have all 4 elements must
have either TATA or DPE, but can have both like
RNApol I promoters, it has upstream control
elements as well
27
Transcription in Eukaryotes
RNA pol II
DPE
DNA
mRNA
TATA box
TFIIB (BRE)
Initiator (Inr)
core promoter (diagrammed above) gives low levels
of transcription upstream elements regulate the
level even further nearby upstream elements are
called proximal promoter elements more distant
upstream elements are called enhancers or
silencers
28
Transcription in Eukaryotes
RNApol III promoter is entirely downstream of the
transcription start contains two 10 bp
sequences, box A and either box B (for tRNA)
or box C (for rRNA)
RNA pol III
box A
DNA
tRNA
box B or C
29
Transcription in Eukaryotes
transcription factor protein that regulates the
transcription of genes general (basal)
transcription factor protein REQUIRED for
transcription often start with TF, like
TFIIB just like with the s factor in
prokaryotes, proteins must bind promoters next,
other TF proteins recognize proteins bound to
promoters RNA polymerase recognizes the cluster
of TF and DNA binding proteins notice the
building up of a machine by protein- protein
inteactions! this is called the pre-initiation
complex RNApol is bound, but not making RNA
30
Transcription in Eukaryotes
once the pre-initiation complex is formed, 2 more
TF factors are needed TFIIE binds and causes RNA
polymerase to be phosphorylated TFIIH binds the
polymerase and acts as a helicase-- unwinds the
DNA so that the phosphorylated RNA polymerase
can make RNA Elongation is very similar-- RNA
polymerase uses AU, TA, GC, CG base
pairing to make the RNA chain from NTPs giving
off PP uses the 3' OH from the message to the
5' phosphate of the NTP One additional
complication RNA polymerase has to have proteins
that unwind nucleosomes-- ie. bacteria don't
have them eukaryotic polymerases have 8-10
proteins, bacteria only 4 some of these
subunits recruit proteins to unwind nucleosomes
31
Transcription in Eukaryotes
Termination is usually caused by recognition of
one of several sequences in the DNA--
different polymerases recognize different
termination sequences ie. RNApol I stops when
a protein binds a particular 18 bp sequence
in the RNA RNApol III stops when it
encounters 6-8 uracils, etc unlike prokaryotes,
eukaryotic polymerases don't seem to stop at
hairpins
32
RNA Processing
newly made RNA molecule is called a primary
transcript-- copied directly from the
DNA before it can serve its eventual function,
RNA must be processed RNA processing includes
being cleaved at specific locations, chemical
modification of some nucleotides, nucleotides
being added, etc. modifications are usually
dependent upon their eventual function ie.
transfer RNAs will have different modifications
than mRNAs just like in transcription,
eukaryotes have more complex RNA processing
33
Ribosomal RNA Processing
70-80 of the total RNA in a cell is ribosomal
RNA (rRNA)
34
Ribosomal RNA Processing
4 different rRNAs distinguished by their
sedimentation coefficients (only 3 in
eukaryotes) 3 of the rRNAs are made by RNApol I
as a single primary transcript RNApol I is
active in the nucleolus, the large dense spot
in the nucleus
transcribed spacers are the parts of the
primary transcript which separate the
rRNAs genome contains multiple copies of the
rRNA primary transcription unit-- needs to
make a lot of rRNA!
35
Ribosomal RNA Processing
transcribed spacers are cut out and then
degraded methyl groups are also added to ribose
hydroxyls and some bases snoRNAs (small
nucleolar RNAs) RNAs that bind to particular
complimentary regions of rRNAs and which also
bind to proteins that methylate the rRNAs
(note the use of complimentary base pairing to
direct these modifications!) methylation of the
rRNA reduces its degradation-- enzyme active
sites don't recognize it because it doesn't have
the hydroxyl groups just like ATP provides the
phosphate group for phosphorylation reactions,
S-adenosyl methionine provides the methyl
group Note the fusion of adenosine (nucleic
acid) and methionine (amino acid)
36
Ribosomal RNA Processing
nearly HALF of the rRNA primary transcript is
transcribed spacer that gets deleted as the
rRNA gets processed, it associates with various
proteins and eventually becomes the large and
small ribosomal subunits Ribosomes are therefore
made in the nucleolus ribosomes also include one
rRNA transcribed by RNApol III this RNA, like
the RNApol I transcript, has multiple copies
arrayed in tandem-- many copies in the same
direction, one right after the other in
genetics, we talk about mechanisms how these
tandem arrays formed
37
Transfer RNA Processing
like the rRNA, tRNA requires extensive removal,
addition and modification of the
nucleotides tRNA RNA molecules that bind to
particular amino acids on one end and
recognize one of the 61 coding codons on the
other these are the ESSENTIAL bridge between
nucleic acids and proteins tRNAs are only about
70-90 nucleotides long and have several hairpin
loops (with complimentary base pairings holding
them together) to form a cloverleaf structure--
in 3D is really more L shaped like the rRNAs,
tRNA is synthesized as a precursor or pre-tRNA
and processed extensively all tRNAs have the
sequence CCA at the 3' end-- some naturally, in
others it is added later
38
Transfer RNA Processing
39
Messenger RNA Processing
prokaryotic mRNA needs little or no processing--
it's ready to go Ribosomes can associate with
prokaryotic mRNA even as it is being
transcribed-- no barrier between mRNA synthesis
and translation
40
Messenger RNA Processing
Eukaryotes require extensive processing of their
mRNAs at the 5' end (ie. first synthesized
part-- start of the transcript) have a 5' cap,
7-methylguanosine is made 'backwards'-- 5' to 5'
linkage to the initial triphospate base added
early after transcription aids in stability and
positioning of the transcript for
translation NOT added by RNA polymerase!
41
Messenger RNA Processing
At the 3' end, most mRNAs contain a 'polyA
tail'-- 50-250 adenosines added by a specific
enzyme, polyA polymerase a signal sequence in
the mRNA directs first the cleavage and then the
addition of the polyA tail 10-35 nucleotides
downstream polyA tail helps to protect the mRNA
from exonucleases and therefore increases its
useful lifespan polyA is recognized by transport
proteins to send it out of the nucleus can also
be used by researchers in the lab to purify
specifically mRNA using a polyT oligonucleotide
42
Messenger RNA Processing
introns sequences in the primary mRNA transcript
that do not appear in the mature mRNA introns
get cut out of the pre-mRNA and the mRNA gets
ligated back together exons regions of the
pre-mRNA or DNA sequence that appear in
proteins introns are present in most protein
coding genes usually found nowadays by comparing
mRNA sequence to genomic DNA RNA splicing
enzymatic process of removing the
introns spliceosome RNA protein complex that
carries out RNA splicing
43
Messenger RNA Processing
most introns start with a 5' GU sequence and end
with a 3' AG introns must also contain an
internal sequence called the branch
point snRNPs small protein RNA complexes that
make up the spliceosome snRNPs bind in 3 parts
U1 binds to the 5' splice site U2 binds to
the branch point U4/U6/U5 brings the ends
of the intron together a spliceosome contains 5
RNAs and 50 proteins-- as big as a ribosome!
U6
U4
U6
U4
U1
U5
U4
U2
U5
U6
U1
U5
U2
U2
U1
44
Messenger RNA Processing
Once snRNPs form a spliceosome, the 5' end is
covalently joined to an adenine nucleotide in
the branch point in a structure called a
lariat once that intermediate is formed, the 3'
end is cleaved, the 2 ends of the exons are
joined together, and the lariat RNA is sent for
degradation splicing occurs during
transcription-- doesn't require
pre-processing we had mentioned ribozymes as RNA
catalysts first ribozymes were self-splicing
intron sequences
45
Messenger RNA Processing
Some introns are NOT degraded after
excision some are involved in rRNA
methylation others can regulate mRNA translation
by complimentary binding to similar sequences
(mRNAs) in other proteins, introns may be left
in or taken out alternative splicing decision
to leave in or take out an intron gives one gene
the ability to make a number of related proteins
using different combinations of potential
introns
46
Messenger RNA Processing
starting with a pre-mRNA
alternative splicing could yield
or
or
or
or
etc. each alternatively spliced transcript would
code for proteins that have overlapping
regions but may have different functions,
locations
47
mRNA Metabolism
most mRNAs aren't around for long-- they are
degraded very rapidly hated by molecular
biologists-- mRNA is hard to work
with half-life average length of time it takes
for 1/2 of the mRNAs to be degraded mRNA
instability allows the cell to regulate gene
expression mRNA also amplifies a DNA
sequence one gene can make many mRNAs, each
making many proteins allows the cell to control
protein levels by controlling how many mRNAs
are made from that gene some promoters are
strong, others weak, others only active sometimes
48
RNA Viruses
RNA viruses use RNA as their primary genetic
material-- ie. HIV these viruses have a very
special enzyme called reverse transcriptase
which can make a DNA copy from the RNA The DNA
copy can then integrate into the host genome
where it then makes RNA that codes for its
proteins and its own genetic material for new
virus particles Molecular biologists use reverse
transcriptase to make 'copy' DNA or cDNA (DNA
made from messenger RNA) allows scientists to
study exactly what mRNA transcripts get made
without having to understand what's happening
with all the splicing, regulatory DNA,
repetitive DNA, etc in the genome
49
RNA Viruses
Reverse transcriptase is different than cellular
enzymes-- it uses RNA to make DNA because it
has a different active site, some nucleotide
analogs can be used to inhibit the reverse
transcriptase active site (competitive
transition state analogs) and make up the most
effective anti-AIDS drugs