1 The information content of genes is in the form of specific sequences of nucleotides along the DNA strands The DNA of an organism leads to specific traits by dictating the synthesis of proteins and of RNA molecules involved in protein synthesis ID: 694382
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Slide1
Chapter 17 – from gene to protein
1
The information content of genes is in the form of specific sequences of nucleotides along the DNA
strands. The
DNA of an organism leads to specific traits by dictating the synthesis of proteins and of RNA molecules involved in protein synthesis (
gene expression
.)
Proteins
are the link between genotype and phenotype
.Slide2
Archibald garrod
The study of metabolic defects provided evidence that genes specify proteins
.
Garrod
discovered
that proteins (enzymes) are the link between genotype and phenotype.
He figured out that some inherited diseases are the
inability to make enzymes
He noticed that the diaper of a baby was very brown. He determined that the baby had
alkaptonuria, which is a recessively inherited disorder where the urine is a brown color. This is due to homogentisic acid which cannot be broken down in the body, so it is excreted in the urine. The reason it cannot be broken down is because there is an absence of the enzyme needed in the biochemical pathway.
2Slide3
Beadle and
tatum
Beadle and Tatum showed the relationship between genes and enzymes
. They used the bread mold
Neurospora
and exposed it to X-rays to get mutants. They found 3 different classes of mutants. Each mutant was defective in a different gene. They exposed these mutants to different environments to see which ones allowed arginine to grow. They deduced that each mutant was unable to carry out one step in the arginine pathway – probably because it lacked the necessary enzyme
3Slide4
One gene – one enzyme
hypothesis…and the evolution of that hypothesis
From Beadle and Tatums experiments, they came up with the one gene, one enzyme hypothesis.
However, not all proteins are enzymes, so it became the one gene- one protein hypothesis
.
BUT…some genes have more than one polypeptide (THINK: quaternary structure of proteins), so it
led to the
one gene- one polypeptide hypothesis
. The newest discoveries have been taken into consideration and the scientific community have updated the definition of a gene as: A gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule.4Slide5
Overview: transcription translation
Transcription = DNA → RNA
Translation = RNA → Protein
DNA
transcription
Primary transcript (pre-mRNA)
RNA processing
mRNA
translation protein
5
Genes provide the instructions for making specific proteins and getting from gene to protein needs two stages: Slide6
Transcription and Translation in prokaryotes vs. eukaryotes
The basic mechanics of transcription and translation are similar in eukaryotes and bacteria.
Bacteria lack nuclei, and their DNA is not separated from ribosomes and other protein-synthesizing equipment.
This allows the coupling of transcription and translation.
In a eukaryotic cell, transcription occurs in the
nucleus
, and translation occurs at
ribosomes
in the cytoplasm.6The molecular chain of command in a cell has a directional flow of genetic information:
DNA RNA proteinFrancis Crick dubbed this concept the central dogma in 1956.Slide7
Coding for amino acids
The message is carried in RNA in the form of
codons
(3 bases). It is read in the 5’ → 3’ direction.
7Slide8
The triplets (codons) code for the specific amino acids
With
a triplet code
, three consecutive bases specify an amino acid, creating 43
(64) possible code words
.
During transcription, one DNA strand, the
template strand
, provides a template for ordering the sequence of nucleotide bases in an mRNA transcript.The mRNA base triplets are called codons. Each codon specifies which one of the 20 amino acids will be incorporated at the corresponding position along a polypeptide chain.The starting point establishes the reading frame
; subsequent codons are read in groups of three nucleotides.8Slide9
Nirenberg determined
the first match: UUU codes for the amino acid
phenylalanine.
Sixty-one
of 64 triplets code for amino
acids.
The
codon
AUG not only codes for the amino acid methionine but also indicates the “start” or initiation of translation.
Three codons do not indicate amino acids but are “stop” signals marking the termination of translation.9
Marshall Nirenberg
deciphered the code for the amino acids in 1961.
The genetic code must have
evolved very early in the history of
life
It is
nearly universal, shared by organisms from the simplest bacteria to the most complex plants and animals
.Slide10
transcription
Promoter
– DNA sequence where RNA attaches and initiates transcription
Terminator
– sequence that signals the end of transcription
Transcription Unit
– sequence of DNA that is transcribed into RNA
DNA → RNA
3 Steps of Transcription: 1. Initiation 2. Elongation 3. Termination
10Slide11
Transcription - Initiation
The
promoter
determines which strand is the
template
and then
transcription factors
help RNA polymerase bind. The TATA box is an important part of the promoter that helps initiate transcription. The transcription complex consists of the promoter, transcription factors, and RNA polymerase.11
RNA polymerase separates the DNA strands at the appropriate point and joins RNA nucleotides complementary to the DNA template strand. Like DNA polymerases, RNA polymerases can assemble a polynucleotide only in its 53 direction (therefore the template strand is 3’
5
’
.)Slide12
Transcription - elongation
The RNA polymerase adds RNA nucleotides about
10-20
at a
time
to the growing 3’ end.
Several mRNA strands can be made at the same time….several different RNA polymerases can all be on the same DNA molecule and can all create
mRNA. This helps the cell make the encoded protein in large amounts. 12Slide13
Transcription - termination
At this point, transcription has given us the primary transcripts or
pre-mRNA
In prokaryotes, termination stops at the termination signal (end of the gene)
In eukaryotes, transcription continues for
10-35
nucleotides past the stop signal. Later in the process, it gets cut down.
13Slide14
RNA Processing:
Modifying the pre-mrna
- At the
5’ end
, a
5’ cap
is added (which is a modified guanine molecule)
At
the 3’ end, there is the poly-A tail (50-250 adenine nucleotides)
functions in helping to inhibit degradation and helps exportation from nucleus)- Both of these modifications have several important functions: Exporting mRNA from the nucleusProtecting mRNA from hydrolytic enzymesHelping the ribosome attach to the 5’ end of the mRNA14
After both ends are modified, the
introns
(non-coding portions) are spliced out. Slide15
RNA Splicing
The introns are cut out using
splicesomes
. Therefore, the mRNA that leaves the nucleus (exons only) is the abridged version that only carries genes – not “filler” DNA.
Introns
= non-coding segments
Exons
= coding segments
15Slide16
RNA splicing technique - splicesomes
There are short sequences at the end of introns that signal to the
snRNP’s
(small nuclear
ribonucleoproteins
). The
snRNP’s
recognize these sites and the splicesomes then cut out the introns and reattach the exons. Ribozyme → RNA molecules that act like enzymes; in some organsisms RNA splicing can occur without additional proteins because the
introns can catalyze their own excision16Slide17
Alternative rna
splicing
Humans can get along with a small number of genes because we can “shuffle” our DNA; different polypeptides can be made depending on which segments we consider introns and which are considered exons.
17Slide18
Translation – from RNA to protein
18
BUILDING A POLYPEPTIDE!Slide19
tRNA -
TRANSLATOR
The cell is always stocked with all 20 AA’s (from diet)
The tRNA is folded like a cloverleaf; on one side it has an
anticodon
that matches up with the codon from the mRNA; on the other side it carries a specific AA
The
tRNA’s
are used over and over; they drop off their AA’s and then go get another to be used again
Wobble → relaxation of 3rd base pairing; sometimes the 3rd base of the ANTICODON has an “I” (inosine), which is an altered adenine; this can match up with U, C, or A; If each anticodon had to be a perfect match to each codon, we would expect to find 61 types of tRNA, but the actual number is about 45, because the anticodons of some tRNAs
recognize more than one
codon (the wobble!!)
CCI anticodon can match up with GGU, GGC and GGA (codons)
19Slide20
Aminoacyl-
tRNA synthetase
This enzyme attaches each AA to its appropriate tRNA.
This process uses 1 ATP
There are 20 different aminoacyl-tRNA synthetases (one for each AA)
Process
:
The active site of the aminoacyl tRNA synthetase binds to the AA and ATP
The ATP loses 2 P groups to become AMP and binds with the AAThen the right tRNA binds to the AA and displaces the AMPThe enzyme then releases the “activated AA”
20Slide21
Ribosomes:
Sites of Translation
Ribosomes consist of 2 subunits, large and small; they are composed of rRNA and proteins
They have 3 binding sites for tRNA:
E
= about to exit
P
= holds the AA chain A = “on-deck” AAThe ribosomes itself catalyzes the peptide bond between amino acids. 21
Like transcription, translation can be divided into 3 stages:
-
initiation
- elongation
- terminationSlide22
Energy source for translation
GTP (
guanosine
triphosphate
) → energy source for translation; this is very similar to ATP and releases energy by breaking off phosphates
22Slide23
Translation - initiation
Steps
:
1. Small ribosomal subunit binds to mRNA leader (5’ end)
2. Initiator tRNA (
methionine
) binds to “start” codon –
AUG
3. Next the large ribosomal subunit binds 4. All of these components (small unit, mRNA, tRNA, large subunit) are brought together by initiation factors and form the translation initiation complex
23Slide24
Translation - elongation
3 step process for each AA:
1. Codon
recognition
2. Peptide
bond formation
3. Translocation
This process uses
elongation factors (proteins)24Slide25
Translation - termination
When a stop
codon (mRNA) gets to get the A-site and instead of a tRNA binding, a
release factor
binds. This adds a
water molecule
to the AA chain, and then releases the chain from the ribosome.
After the chain is released, all the factors dissociate from one another.
25Slide26
Overview – protein synthesis
26Slide27
Polyribosomes
or polysomes
This is when many ribosomes trail along the same mRNA molecule. They can translate many proteins simultaneously and therefore are much more efficient.
27Slide28
Post-translational modifications
During and after synthesis, a polypeptide spontaneously
coils and folds to its three-dimensional shape.In addition, proteins may require post-translational modifications
before doing their particular job.These modifications may require additions such as sugars, lipids, or phosphate groups to amino acids.In other cases, a polypeptide may be cleaved in two or more pieces OR two or more polypeptides may join to form a protein with quaternary structure
.
28Slide29
Signal peptides – determine whether ribosome will be attached or free
All ribosomes start as free (in the cytosol
); however, the polypeptide can cue the ribosome to go attach to the ER and become bound. The
signal peptide
is a sequence of
about 20 AA’s
near the front of the strand that tells the ribosome to go attach. This is the case for proteins/ enzymes that
are going to be secreted from the cell
. The signal recognition particle (SRP) sees this signal peptide and brings the ribosome to the ER to attach.
29Slide30
mutations
A
point
mutation (also called a
substitution
)
is a change in
one base pair
. It can have huge effects (sickle cell) or no affect at all (silent mutation), depending on which base is affected and where the AA is located in the protein. 30Mutations are changes in the genetic material of a cell (or virus). They are
the ultimate source of new genes (and genetic diversity!)Slide31
Missense
and nonsense mutations
Missense
= codes for a different AA
Nonsense
= changes into a stop codon, so it leads to a nonfunctional
protein
Silent
= changes the nucleotide but it codes for the same AA
31Slide32
Frameshift mutations
A
frameshift
mutation is when there is an
insertion
or
deletion
that causes the reading frame to change. This means that all of the AA’s after the mutation will be wrong. It has disastrous effects.
32Slide33
mutations can occur during DNA replication, DNA repair, or DNA
recombination
Errors during DNA replication or recombination can lead to nucleotide-pair substitutions, insertions, or deletions.
Mutagens are chemical or physical agents that interact with DNA to cause mutations.Physical agents include high-energy radiation like X-rays and ultraviolet light.
Chemical mutagens cause mutations in different ways.
33