From DNA to Protein: Genotype to Phenotype
From DNA to Protein: Genotype to Phenotype
One Gene, One Polypeptide
DNA, RNA, and the Flow of Information
Transcription: DNA-Directed RNA Synthesis
The Genetic Code
Preparation for ________ Linking RNAs, Amino Acids, and Ribosomes
Translation: RNA-Directed Polypeptide Synthesis
Regulation of Translation
Posttranslational Events
Mutations: ________ Changes in Genes
One Gene, One Polypeptide
A gene is defined as a DNA sequence.
There are many steps between genotype and phenotype; genes cannot by
themselves produce a phenotype.
One Gene, One Polypeptide
In the 1940s, Beadle and Tatum showed that when an altered gene resulted in an
altered phenotype, that altered phenotype always showed up as an altered enzyme.
They experimented with strains of the bread mold Neurospora: a wild-type, and
several mutant strains.
Their results suggested that mutations cause a defect in only one enzyme in a
metabolic pathway.
This lead to the one-gene, one-enzyme hypothesis.
One Gene, One Polypeptide
The geneenzyme connection has undergone several modifications. Some enzymes
are composed of different ________ coded for by separate genes.
This suggests, instead of the one-gene, one enzyme hypothesis, a one-gene,
one-polypeptide relationship.
DNA, RNA, and the Flow of Information
The expression of a gene takes place in two steps:
ง ________ makes a single-stranded RNA copy of a segment of the DNA.
ง ________ uses information encoded in the RNA to make a polypeptide.
DNA, RNA, and the Flow of Information
RNA (ribonucleic acid) differs from DNA in three ways:
ง RNA consists of only one polynucleotide strand.
ง The sugar in RNA is ________ not deoxyribose.
ง RNA has ________ instead of thymine.
RNA can base-pair with single-stranded DNA (adenine pairs with uracil instead
of thymine) and also can fold over and base-pair with itself.
DNA, RNA, and the Flow of Information
Francis Crick's ________ ________ stated that DNA codes for RNA, and RNA codes
for protein.
How does information get from the nucleus to the cytoplasm?
What is the relationship between a specific nucleotide sequence in DNA and a
specific amino acid sequence in protein?
DNA, RNA, and the Flow of Information
Messenger RNA, or mRNA moves from the nucleus of ________ cells into the
cytoplasm, where it serves as a template for protein synthesis.
Transfer RNA, or tRNA, is the link between the code of the mRNA and the amino
acids of the polypeptide, specifying the correct amino acid sequence in a
protein.
DNA, RNA, and the Flow of Information
Certain viruses use RNA rather than DNA as their information molecule during
transmission.
These viruses transcribe from RNA to RNA; they make a complementary RNA strand
and then use this "opposite" strand to make multiple copies of the viral genome
by transcription.
HIV and certain tumor viruses (called retroviruses) have RNA as their
infectious information molecule; they convert it to a DNA copy inside the host
cell and then use it to make more RNA.
Transcription: DNA-Directed RNA Synthesis
In normal prokaryotic and eukaryotic cells, transcription requires the
following:
ง A DNA template for ________ base pairing
ง The appropriate ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) to act
as substrates
ง The enzyme RNA polymerase
Transcription: DNA-Directed RNA Synthesis
Just one DNA strand (the template strand) is used to make the RNA.
For different genes in the same DNA molecule, the roles of these strands may
be reversed.
The DNA double helix partly unwinds to serve as template.
As the RNA transcript forms, it peels away, allowing the already transcribed
DNA to be rewound into the double helix.
Transcription: DNA-Directed RNA Synthesis
The first step of transcription, ________ begins at a promoter, a special
sequence of DNA.
There is at least one promoter for each gene to be transcribed.
The RNA polymerase binds to the promoter region when conditions allow.
The promoter sequence directs the RNA polymerase as to which of the double
strands is the template and in what direction the RNA polymerase should move.
Transcription: DNA-Directed RNA Synthesis
After binding, RNA polymerase unwinds the DNA about 20 base pairs at a time
and reads the template in the 3ข-to-5ข direction (elongation).
The new RNA ________ from its 5ข end to its 3ข end; thus the RNA transcript is
antiparallel to the DNA template strand.
Transcription errors for RNA polymerases are high relative to DNA polymerases.
Transcription: DNA-Directed RNA Synthesis
Particular base sequences in the DNA specify termination.
Gene mechanisms for termination vary:
ง For some, the newly formed transcript simply falls away from the DNA template.
ง For other genes, a helper protein pulls the transcript away.
ง In prokaryotes, translation of the mRNA often begins before transcription is
complete.
The Genetic Code
A genetic code relates genes (DNA) to mRNA and mRNA to the amino acids of
proteins.
mRNA is read in ________ segments called codons.
The number of different codons possible is 64 (43), because each position in
the codon can be occupied by one of four different bases.
The 64 possible codons code for only 20 amino acids and the start and stop
signals.
The Genetic Code
AUG, which codes for methionine, is called the start codon, the initiation
signal for translation.
Three codons (UAA, UAG, and UGA) are stop codons, which direct the ribosomes
to end translation.
The Genetic Code
After subtracting start and stop codons, the remaining 60 codons code for 19
different amino acids.
This means that many amino acids have more than one codon. Thus the code is
redundant.
However, the code is not ambiguous. Each codon is assigned only one amino
acid.
The Genetic Code
In the early 1960s, molecular biologists broke the genetic code.
Nirenberg prepared an artificial mRNA in which all bases were uracil (poly U).
When incubated with additional components, the poly U mRNA led to synthesis of
a polypeptide chain consisting only of phenylalanine amino acids.
UUU appeared to be the codon for phenylalanine.
Other codons were deciphered from this starting point.
Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
The molecule tRNA is required to assure specificity in the translation of mRNA
into proteins.
The tRNAs must read mRNA correctly.
The tRNAs must carry the correct amino acids.
Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
The codon in mRNA and the amino acid in a protein are related by way of an
adaptera specific tRNA molecule.
tRNA has three functions:
ง It carries an amino acid.
ง It associates with mRNA molecules.
ง It interacts with ribosomes.
Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
A tRNA molecule has 75 to 80 nucleotides and a ________ shape (conformation).
The shape is maintained by complementary base pairing and hydrogen bonding.
The three-dimensional shape of the tRNAs allows them to combine with the
binding sites of the ribosome.
Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
At the 3ข end of every tRNA molecule is a site to which its specific ________
________ binds covalently.
Midpoint in the sequence are three bases called the anticodon.
The anticodon is the contact point between the tRNA and the mRNA.
The anticodon is complementary (and antiparallel) to the mRNA codon.
The codon and anticodon unite by complementary base pairing.
Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
Amino acids are attached to the correct tRNAs by activating enzymes called
aminoacyl-tRNA synthetases.
The enzyme has a three-part active site that binds:
ง A specific amino acid
ง ATP
ง A specific tRNA, charged with a high-energy bond
The high-energy bond provides the energy for making the peptide bond.
Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
The reactions have two steps:
ง Enzyme + ATP + AA ฎ enzymeAMPAA + PPi
ง EnzymeAMPAA + tRNA ฎ enzyme + AMP + tRNAAA
Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
Each ribosome has two subunits: a large one and a small one.
In eukaryotes the large one has three different associated rRNA molecules and
45 different proteins.
The small subunit has one rRNA and 33 different protein molecules.
When they are not translating, the two subunits are separate.
Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
The proteins and rRNAs are held together by ionic bonds and hydrophobic
forces.
The large subunit has four binding sites:
ง The T site where the tRNA first lands
ง The A site where the tRNA anticodon binds to the mRNA codon
ง The P site where the tRNA adds its amino acid to the polypeptide chain
ง The E site where the tRNA goes before leaving the ribosome
Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
The small ribosomal subunit plays a role in validating the three-base-pair
match between the mRNA and the tRNA.
If hydrogen bonds have not formed between all three base pairs, the tRNA is
ejected from the ribosome.
Translation: RNA-Directed Polypeptide Synthesis
Translation begins with an initiation complex: a charged tRNA with its amino
acid and a small subunit, both bound to the mRNA.
This complex is bound to a region upstream of where the actual reading of the
mRNA begins.
The start codon (AUG) designates the first amino acid in all proteins.
The large subunit then joins the complex.
The process is directed by proteins called initiation factors.
Translation: RNA-Directed Polypeptide Synthesis
Ribosomes move in the 5ข-to-3ข direction on the mRNA.
The peptide forms in the NtoC direction.
The large subunit catalyzes two reactions:
ง Breaking the bond between the tRNA in the P site and its amino acid
ง Peptide bond formation between this amino acid and the one attached to the
tRNA in the A site
This is called peptidyl transferase activity.
Translation: RNA-Directed Polypeptide Synthesis
After the first tRNA releases ________ it dissociates from the ribosome and
returns to the cytosol.
The second tRNA, now bearing a dipeptide, moves to the P site.
The next charged tRNA enters the open A site.
The peptide chain is then transferred to the P site.
These steps are assisted by proteins called elongation factors.
Translation: RNA-Directed Polypeptide Synthesis
When a stop codonUAA, UAG, or UGAenters the A site, a release factor and a
water molecule enter the A site, instead of an amino acid.
The newly completed protein then separates from the ribosome.
Regulation of Translation
Antibiotics are defensive molecules produced by some fungi and bacteria, which
often destroy other microbes.
Some antibiotics work by blocking the synthesis of the bacterial cell walls,
others by inhibiting protein synthesis at various points.
Because of differences between prokaryotic and eukaryotic ribosomes, the human
ribosomes are unaffected.
Regulation of Translation
Polysomes are mRNA molecules with more than one ribosome attached.
These make protein more rapidly, producing multiple copies of protein
simultaneously.
Posttranslational Events
Two posttranslational events can occur after the polypeptide has been
synthesized:
ง The polypeptide may be moved to another location in the cell, or secreted.
ง The polypeptide may be modified by the addition of chemical groups, folding,
or trimming.
Posttranslational Events
As the polypeptide chain forms, it folds into its 3-D shape.
The amino acid sequence also contains an "address label" indicating where in
the cell the polypeptide belongs. It gives one of two sets of instructions:
ง Finish translation and be released to the cytoplasm.
ง Stall translation, go to the ER, and finish synthesis at the ER surface.
Posttranslational Events
Polypeptides sent to the cytoplasm may contain information (signal sequences)
that specifies a destination.
The signal sequence binds to ________ proteins at the outer membrane of the
appropriate organelle.
A channel opens in the membrane, allowing the protein to pass through.
In the process, the protein usually is unfolded by a ________ so that it can
pass through the channel.
Posttranslational Events
Polypeptides destined for the ER have a 25-amino-acid-long leader sequence.
Before translation is finished, the leader sequence binds to a signal
recognition particle.
This stalls protein synthesis until the ribosome attaches to a specific
receptor protein on the surface of the ER.
Translation continues with the protein moving through a pore in the ER
membrane.
Posttranslational Events
Other signals are needed to direct further protein sorting:
ง Sequences of amino acids that allow the protein to stay in the ER
ง Sugars added in the Golgi apparatus to form glycoproteins, which go to
lysosomes or the plasma membrane
Proteins with no signals from the ER go through the Golgi apparatus and are
secreted from the cell.
Posttranslational Events
Most proteins are ________ after translation.
These modifications are often essential to the functioning of the protein.
Three types of modifications:
ง Proteolysis (cleaving)
ง Glycosylation (adding sugars)
ง Phosphorylation (adding phosphate groups)
Mutations: Heritable Changes in Genes
Mutations are heritable changes in DNAchanges that are passed on to daughter
cells.
Multicellular organisms have two types of mutations:
ง Somatic mutations are passed on during mitosis, but not to subsequent
generations.
ง Germ-line mutations are mutations that occur in cells that give rise to
gametes.
Mutations: ________ Changes in Genes
Some mutations, called conditional mutants, exert their effect only under
certain restrictive conditions.
A temperature-sensitive mutant allele, for example, may code for an enzyme
that is altered at the restrictive temperature.
Mutations: Heritable Changes in Genes
All mutations are alterations of the DNA nucleotide sequence and are of two
types:
ง Point mutations are mutations of single genes.
ง Chromosomal mutations are changes in the arrangements of chromosomal DNA
segments.
Mutations: Heritable Changes in Genes
Point mutations result from the addition or subtraction of a base or the
substitution of one base for another.
Point mutations can occur as a result of mistakes during DNA replication or
can be caused by environmental mutagens.
Because of redundancy in the genetic code, some point mutations, called silent
mutations, result in no change in the amino acids in the protein.
Mutations: Heritable Changes in Genes
Some mutations, called missense mutations, cause an amino acid substitution.
An example in humans is sickle-cell anemia, a defect in the b-globin subunits
of hemoglobin.
The b-globin in sickle-cell differs from the normal by only one amino acid.
Missense mutations may reduce the functioning of a protein or disable it
completely.
Mutations: Heritable Changes in Genes
Nonsense mutations are base substitutions that substitute a stop codon.
The shortened proteins are usually not functional.
Mutations: Heritable Changes in Genes
A ________ ________ consists of the insertion or deletion of a single base in
a gene.
This type of mutation shifts the code, changing many of the codons to
different codons.
These shifts almost always lead to the production of nonfunctional proteins.
Mutations: Heritable Changes in Genes
DNA molecules can break and re-form, causing four different types of
mutations:
ง Deletions are a loss of a chromosomal segment.
ง Duplications are a repeat of a segment.
ง Inversions result from breaking and rejoining when segments get reattached in
the opposite orientation.
ง Translocations result when a portion of one chromosome attaches to another.
Mutations: Heritable Changes in Genes
Spontaneous mutations are permanent changes, caused by any of several
mechanisms:
ง Nucleotides occasionally change their structure (called a tautomeric shift).
ง Bases may change because of a chemical reaction.
ง DNA polymerase sometimes makes errors in replication which can escape being
repaired.
ง Meiosis is imperfect. Nondisjunction and translocations can occur.
Mutations: Heritable Changes in Genes
Induced mutations are permanent changes caused by some outside agent
(mutagen).
Mutagens can alter DNA in several ways:
ง Altering covalent bonds in nucleotides
ง Adding groups to the bases
ง Radiation damages DNA:
q Ionizing radiation (X rays) produces free radicals.
q Ultraviolet radiation is absorbed by thymine and causes interbase covalent
bonds to form.
Mutations: Heritable Changes in Genes
Mutations have both benefits and costs.
Germ line mutations provide genetic diversity for evolution, but usually
produce an organism that does poorly in its environment.
Somatic mutations do not affect offspring, but can cause cancer.
Mutations: Heritable Changes in Genes
Mutations are rare events and most of them are point mutations involving one
nucleotide.
Different organisms vary in mutation frequency.
Mutations can be detrimental, neutral, or occasionally beneficial.
Random accumulation of mutations in the extra copies of genes can lead to the
production of new useful proteins.
Video 12-01
Animation 12.1 Transcription
Animation 12.2 Deciphering the Genetic Code
Animation 12.3 Protein Synthesis