Transcript From Gene to Protein

From Gene to Protein
Campbell and Reece
Chapter 17
Gene Expression
process by which DNA directs the
synthesis of proteins or RNA
 synthesis of proteins

1.
2.
transcription
translation
How Gene to Protein Figured
Out
Evidence from study of metabolic
disorders:
 1902: British physician 1st to suggest
genes responsible for phenotype
thru enzymes that catalyze specific
chem rx in the cell

Inborn Errors of Metabolism
Garrod hypothesized that symptoms
of an inherited disease are due to a
gene that leads to inability to make a
certain enzyme
 1 of 1st to realize Mendel’s principle’s
of heredity applied to more than pea
plants

Alkaptonuria

Signs & Symptoms:
urine turns black when alkapton
(chemical in urine) reacts with air
 missing enzyme in pathway that
degrades phenylalanine (a.a.)

Beadle & Tatum Experiment
worked with a bread mold Neurospora
crassa
 bombarded it with radiation (already
known to cause genetic changes)
 then checked for survivors who had
different nutritional needs from wild-type
mold

Beadle & Tatum Experiment
individually put yeast in different
mediums (agar with different nutrients)
 identified mutants that could not survive
on minimal nutrients  placed them in
complete growth medium (minimal med.
+ all 20 a.a. + few vitamins & minerals)

1-Gene-1-Polypeptide
Beadle & Tatum’s results supported their
hypothesis
 1958: Nobel prize

1-Gene-1-Polypeptide

revised over time:
not all proteins are enzymes
 some proteins have >1 polypeptide
 now: 1- gene-1-protein hypothesis
 not 100%: some eukaryotic genes can each
code for a set of closely related polypeptides
via alternative splicing

Transcription: short version
the synthesis of RNA using information in
DNA
 mRNA made using complimentary base
pairing

Translation: short version
synthesis of a polypeptide using the
information in mRNA
 “translates” message in mRNA  a.a.

The Genetic Code
4 nucleotide bases to code for 20 a.a.
 triplet code: 3 consecutive bases code for 1
of the a.a./ stop

Template Strand

during transcription:
DNA helix unwound
 1 strand only transcribed (could be either side
depending on the gene)

mRNA
uracil added as compliment to adenine
 ribose as its 5-carbon sugar
 single stranded

Codons
nucleotide triplets of DNA or mRNA that
specifies a particular amino acid or
termination signal
 basic unit of the genetic code
 written in 5’  3’ direction (in DNA 3
bases read in 3’  5’ direction)

Genetic Code
Cracking the Code
early 1960’s
 Nirenberg: synthesized mRNA using only
uracil (UUUUUUU…)

added it to test tube with all 20 a.a., ribosomes
 translated into polypeptide made up of
phenyalanine
 now knew UUU = Phe
 did same for AAA= Lys, CCC = Pro, GGG = Gly

Cracking the Code
all 64 a.a. deciphered by mid-1960’s
 3 codons code for “stop” marking end of
translation
 AUG functions as “start” & Met


Met may or may not be clipped off later
Genetic Code is Redundant
>1 triplet codes for each of the a.a. but any
1 triplet codes for only 1 a.a
 redundant triplets usually only differ in
the 3rd base

Reading Frame
translating the code in correct groupings
 example:
Did the red dog eat the bug?
Idt her edd oge att heb ug?

Reading Frame
Evolution of Genetic Code

code is nearly universal:
bacteria  complex multicellular organisms
CAU = His
 insert genes into other species & get same
result (human insulin gene in bacteria)


exceptions: certain unicellular eukaryotes
& in organelle genes of some species
RNA Polymerase
unwinds 2 strands of DNA
 binds nucleotides together as build mRNA


only in 5’  3’ direction (like DNA polymerase)
3 Stages of Transcription
1.
2.
3.
Initiation
Elongation
Termination
Initiation

After RNA polymerase binds to promoter,
¤ DNA strands unwind

polymerase begins RNA synthesis @ start pt.
on template strand
Initiation
promoter: usually includes w/in it the
transcription start point (a nucleotide
where transcription begins) & extends
several dozen or more nucleotide pairs
upstream from start pt.
 RNAP can assemble nucleotides only in 5’
 3’ direction (just like DNA polymerase)
 unlike DNAP, RNAP does not require a
primer

Start Point
nucleotide where RNA synthesis actually
begins
 RNAP binds in precise location &
orientation on the promoter  where
determines where transcription starts &
which of the 2 strands will be transcribed

RNA Polymerase

Bacteria:


1 single RNAP used to make all types RNA
Eukaryotic Cells:
@ least 3 types RNA polymerase
 II used for RNA synthesis
 I and III used to transcribe RNA not used for
protein synthesis

RNA Polymerase

Prokaryotes :


RNAP recognizes & binds to the promoter by
itself
Eukayotes:

collection of proteins , transcription factors,
mediate the binding of RNAP & initiation of
transcription
Transcription Factors
must 1st attach to promoter b/4 RNAP II
can bind to it
 RNAP II + transcription factors =
Transcription Initiation Complex
 TATA box: DNA sequence in eukaryotic
promoters crucial in forming the
transcription initiation complex

Elongation
RNAP moves downstrean, unwinding the
DNA & elongating the RNA transcript 5’ 
3’
 ~ 10 – 20 nucleotides exposed
 in wake of transcription the 2 DNA strands
spontaneously rewind
 length of DNA transcribed = transcription
unit

Elongation
Termination
mechanism differs between prokaryotes &
eukaryotes
 Bacteria: transcription proceeds thru
terminator sequence in the DNA  the
transcribed RNA functions as the
terminator sequence  causing RNAP to
detach
 prokaryotes have no further modification

Termination in Eukaryotes
RNAP II transcribes a portion of DNA
called the polyadenylation signal
(AAUAAA) in the pre-mRNA
 ~10 – 35 nucleotides downstream from
that sequence proteins ass’c with
transcription cut the pre-mRNA free from
the polymerase
 pre-mRNA then  modified

RNA Processing
in eukaryotes only
 both ends of primary transcript altered
 certain interior sections cut out &
remaining parts spliced back together

mRNA Ends
5’ end receives a 5’cap: modified G is
added after ~ 20 – 40 nucleotides in
mRNA
 3’ end modified: enzyme adds 50 -250 A’s
to the AAUAAA forming a poly-A tail

Functions of Modified Ends of
mRNA
1.
2.
3.
facilitate exit of mRNA from nucleus
protect mRNA from degradation of
hydrolytic enzymes
help ribosomes attach to the 5’ end
RNA Splicing
cut-and-paste job removing segments of
RNA that were transcribed
 average size transcript: 27,000 nucleotides
 average size protein: 1,200 nucleotides
(400 a.a.)

Introns

noncoding, intervening sequence w/in
primary transcript that is removed from
the transcript during RNA processing; also
refers to the region of DNA from which
this sequence was transcribed
Exons

sequence w/in primary transcript that
remains in the RNA after RNA processing;
also refers to the region of DNA from
which this sequence was transcribed
RNA Splicing
signal: short nucleotide sequence @ each
end of an intron
 particle called “snurp” recognizes splice
sites

small nuclear ribonucleoproteins (snRNP’s)
 in nucleus
 made of RNA + protein
 small nuclear RNA ~150 nucleotides

Spliceosome
combination of several different snRNP’s
(almost size of ribosome)
 interact with certain sites along intron
releasing intron  rapidly degraded
 then joins ends of exons together

RNA Splicing
Ribozymes


RNA molecules that
function like enzymes
in some organisms
intron RNA can act
like ribozyme &
catalyze its own
excision
Ribozymes
3 properties of RNA enables some RNA
molecules to function as enzymes:
1. single-stranded: 1 sequence can interact
w/another using base pairing
2. some of bases contain functional groups
(like a.a) that could participate in
catalysis
3. ability to form H-bonds adds specificity

RNA
Importance of Introns
still having debate about importance of
introns & RNA splicing in evolution
 they both have adaptive benefits
 do not know functions of most introns

Importance of Introns
single gene can encode >1 kind of
polypeptide
 know many genes that make 2 or more
different polypeptides depending on what
was removed as introns during gene
splicing
 called: alternative RNA splicing

Alternative RNA Splicing


Drosophila sex
differences due to
how RNA transcript is
spliced
Human Genome
Project: 1 of reasons
humans get by with
same # genes as a
nematode
Translation: Closer Look
tRNA: transfers a.a. from cytoplasmic pool
of a.a to ribosome where it’s a.a. is added
to polypeptide chain
 cell keeps supply of all 20 a.a. on hand

degradation of other molecules
 synthesizes them using building blocks in
cytoplasm

tRNA
brings specific a.a to ribosome
 1 end has a.a./ other end has anticodon
which H-bonds with codon on ribosome
 tRNA translates the codes into the
corresponding a.a.
 tRNA is transcribed from DNA templates
& used repeatedly
 tRNA made of ~80 nucleotides long with
some regions folded back on self due to
base pairing

tRNA Structure
tRNA Structure


3’ end: a.a. attached
opposite end:
anticodon
Accurate Translation

1.
requires 2 instances of molecular
recognition:
tRNA that binds to particular a.a.

2.
correct match made by group enzymes called
aminoacyl-tRNA synthetases: their
active site fits only 1 of the 20 a.a.
pairing of tRNA anticodon with mRNA
codon
tRNA Wobble

~ 45 different ones (not 61 like genetic
code would suggest)

possible because pairing the 3rd base of codon
& 3rd base of anticodon: relaxed base pair
rules
U can pair with A or G in 3’ end of codon (3rd
position)
 called a “wobble”

Ribosomes

subunits made in nucleolus
rRNA transcribed & added to proteins
imported from cytoplasm
 ribosomal subunits  cytoplasm, join only
when translating mRNA
 subunits ~1/3 protein & 2/3 rRNA

bacteria: 3 molecules rRNA
 eukaryotes: 4 molecules rRNA

Ribosome Structure
eukaryotic ribosomes slightly larger than
prokaryotic ones
 pharmaceutical products (antibiotics)
designed to inactivate bacterial ribosomes
that have no effect on ours

Tetracyclines
 Streptomycin

Ribosome Structure
4 binding sites: (1st for mRNA, others for
tRNA)
1. mRNA binding site
2. P site: peptidyl-tRNA holds the tRNA
carrying the growing polypeptide chain
3. A site: aminoacyl-tRNA holds tRNA
carrying next a.a to be added
4. E site: exit, where discharged tRNAs
leave ribosome

Ribosome
holds tRNA & mRNA in close proximity &
catalyzes the formation of new peptide
bond holding the 2 a.a together adding to
carboxyl end of last a.a. in growing
polypeptide chain
 peptide chain passes thru exit tunnel in
large subunit as it grows longer

Translation
3 Stages:
1. Initiation
2. Elongation
3. Termination

Initiation
small ribosomal subunit attaches to
mRNA
 downstream from this attachment is the
start codon AUG
 tRNA with UAC (Met) binds to it
 large ribosomal subunit attaches (1GTP)
 initiation factors (proteins) required to
bring it all together

Elongation
1.
Codon recognition


2.
Peptide bond formation


3.
anticodon of incoming tRNA w/c’ base
1 GTP increases accuracy & efficiency
part of rRNA catalyzes reaction
amino end of newest a.a + carboxyl end of peptide
chain transferring pep. chain to tRNA @ A site
Translocation


ribosome moves so tRNA @ A site  P site
1 GTP
Termination
1.
2.
3.
ribosome reaches stop codon the A site
accepts a “release factor” (shaped like
tRNA but does not have aminoacyl part)
promotes release of bond between P site,
mRNA, & last tRNA
2 ribosomal subunits & ass’c proteins
come apart
Animation Time!
http://bcs.whfreeman.com/thelifewire/co
ntent/chp12/1202003.html
 http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf:
:535::535::/sites/dl/free/0072437316/120
077/micro06.swf::Protein%20Synthesis

Try at home: interactive

http://www.wiley.com/college/boyer/047
0003790/animations/translation/translat
ion.htm
Polyribosomes
1 ribosome can make polypeptide of
average size: 1 min
 typically many ribosomes are translating a
single mRNA @ given time
 1st ribosome gets far enough past start
codon 2nd ribosome can get started
 allow cell to make many copies of
polypeptide very quickly

Polyribosomes
Primary Structure
as polypeptide chain grows longer from
ribosome it will spontaneously start to fold
& coil as result of a.a side chain
interactions
 genes determine 1’ structure which then
determines 2’, 3’ and 4’ structures

Chaperonins

proteins that help with the folding
Post-Translational Modifications

additional steps that may be required b/4
protein can do its job
attachment of sugars, lipids, phosphate
groups to a.a
 enzymatic removal of 1 or more a.a. from
leading end (amino end)

Modification of Insulin
Targeting Polypeptides  Specific
Locations in Cell
free ribosomes make proteins used in
cytoplasm
 bound ribosomes (RER) attached to
cytosolic side while polypeptide being
released into endomembrane system
 both have identical small & large subunits

Ribosomes
Signal Peptide
growing polypeptide cues ribosome to
attach to ER
 polypeptides of proteins destined for
endomembrane system have signal
peptide: sequence of ~20 a.a. at or near
leading end (N-terminus) is recognized by
a protein-RNA complex called signalrecognition particle or SRP

SRP
escorts ribosome to receptor protein on
ER membrane
 receptor part of multiprotein translocation
complex
 ribosome continues to make polypeptide
which enters ER thru protein pore
 signal protein usually removed by enzyme

Proteins  Organelles
use other signal peptides for protein
destined for chloroplast, mitochondria, or
interior of nucleus
 in these, proteins made in cytosol then 
to organelle
 signal proteins target or “address” proteins
for secretion or to cellular locations


used by prokaryotes too
Mutations

ultimate source of new genes

large scale mutations


chromosomal rearrangements: chap. 15
small scale mutations

1 or a few nucleotide bases changed
Try @ Home

http://www.bodrum-hotels.com/genemutations/gene-mutations-and-proteinsworksheet.html
Point Mutations
changes in single nucleotide pair
 if occurs in gamete or cell that  gamete
will be passed on to offspring
 if mutation has adverse effect on
phenotype is called a genetic disorder or
hereditary disease
 if mutation causes organism to die before
fully developed it is said to be lethal
 if mutation results in no change in
phenotype is said to be silent

Sickle Cell Anemia
Familial Cardiomyopathy



point mutation
dominant
possible cause of
sudden death of
young athletes
Substitutions
replacement of 1 nucleotide pair by another
pair: a few will improve activity of protein it
is coding for but most will be detrimental
 some silent due to redundancy of genetic
code
 if changes 1 a.a. for another called missense
mutation



if substituted a.a. similar to real one no effect
some substitutions will have major consequences
Nucleotide-Pair Substitutions
Nucleotide-Pair Substitution
1.
2.
Silent
Missense:

3.
most substitutions in this category
Nonsense: substitution changes from 1
a.a.  stop codon


resulting polypeptide is shorter
nearly all  nonfunctional proteins
Insertions & Deletions
(+) or (-) of nucleotide pairs in a gene
 disastrous effects
 may alter reading frame  triplet codon
shifts on mRNA
 called frameshift mutation



whenever insertion or deletion not in a multiple of 3
if not causes major missense
Mutagens
any chemical or physical agent that
interacts with DNA & can cause a
mutation
 1920’s: Muller used x-rays to make mutant
Drosophila & he discovered it does same
in humans
 mutagenic radiation includes:


UV radiation cause thymine dimers in DNA
Thymine Dimers
Chemical Mutagens

nucleotide analogs


similar to normal DNA nucleotides
insert self into DNA
Chemical Mutagens

some cause chemical changes in bases that
changes their pairing properties
How Mutagens Determined
Gene Expression in 3 Domains
Differences



some in gene expression among eubacteria,
archaea, and eukaryotes
if no nucleus: translation can begin b/4
transcription is over
Archaea show similarities to Eubacteria and
eukaryotes in processes of gene expression
What is a Gene?

region of DNA whose final functional
product is either a polypeptide or an ENA
molecule