Transcript Slide 1

FROM GENE TO PROTEIN
Proteins make the Organism
Chapter 17
Overview of
the Gene to
Protein
Concept
•The Triplet Code
The Dictionary of The Genetic Code
Transcription
• Genes have nucleotide segments upstream of
them called promoters
• Promoter sites have a segment that are high in
Thymine and Adenine residues – called TATA
boxes
• Certain specific transcription factors bind to
TATA box of the promoter site
• RNA Polymerase can then recognize the
promoter site and transcription can begin
• RNA Pol. Uses ATP, UTP, GTP and CTP to
copy the template (anti-sense) strand of the
gene.
Transcription, cont’d.
• Initiation (Binding of RNA Polymerase)
• Elongation (Lengthening of mRNA strand)
• Termination (End of transcription)
Transcription
Initiation
Enhancers and Silencers
• Enhancers are sequences further
upstream of the gene’s promoter
• Special proteins called Activators bind to
these enhancer sequences to increase
rate of transcription
• Silencer sequences are also found
upstream of gene promoters
• Proteins called Repressors ind to silencers
and decrease rate of transcription
mRNA Elongation
Pre-mRNA Processing
• Splicing of introns (By
snRNPS – spliceosomes)
• 5’ Methylguanosine cap
• 3’ Poly A tail
mRNA SPLICING
Spliceosomes
Are introns necessary?
• Humans have just 20,000 to 25,000 genes, but
the human body may contain more than 2 million
different proteins, each having different
functions.
• One gene one protein? Can’t be!
• A bacterial chromosome contains between 5001500 genes (depending on type of bacterium)
and about the same number of proteins
• Eukaryotes have many introns, whereas bacteria
have few to none
Alternate Splicing of Exons
• A classic example of alternate splicing is the rat muscle
protein, troponin T. The gene consists of five exons,
each representing a domain of a final protein. These
exons are each separated by an intron. The five exons
are W, X, Alpha, Beta, and Z. Two types of protein are
found. The alpha form consists of exons W, X, alpha and
Z. The beta form consists of the W, X, Beta and Z exons.
The two different types of the protein are produced by
alternative splicing of the same gene. The two different
gene products are produced by selective splicing such
that introns three and four and the fourth exon are
spliced as one unit. In some manner the 5' GT sequence
of intron 3 and the 3' AG sequence of the fourth intron
are used during the splcing event.
Introns Early or Late?
• The Introns-early theory says exons were minigenes. At
some stage, such as precellular life, minigenes would
have functioned as genes do today. At a later stage in
evolution, the minigenes were assembled to make whole
genes. Introns were the functionless pieces that held the
exons together. All genes were built that way. Bacteria
have no introns, and single-celled eukaryotes have very
few because they lost them in later evolutionary stages.
That's the introns-early theory.
• Meanwhile, the introns-late theory also has a hard time
explaining the usefulness of introns. One possibility is,
"Introns originated to circumvent the problem of the
random distribution of stop codons in random primordial
sequences"
Capping and Polyadenylation
•In eukaryotes, the RNA is processed at both ends before it is spliced.
• At the 5' end, a cap is added consisting of a modified GTP (guanosine
triphosphate). This occurs at the beginning of transcription. The 5' cap is
used as a recognition signal for ribosomes to bind to the mRNA. At the 3'
end, a poly(A) tail of 150 or more adenine nucleotides is added. The tail
plays a role in the stability of the mRNA.
• The cap and tail also protect the mRNA from degradation.
Translation
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Ribosomes
mRNA
tRNA, tRNA-acyl synthetase
Amino Acids
•Initiation
•Elongation
•Termination
Ribosomes
•Composed of rRNA
and proteins
•Large and small
subunits lock
together to translate
mRNA
The Nucleolus – Home of
Ribosome Assembly
The Nucleolus – Home of
Ribosome Assembly
• The nucleolus is located inside the nucleus. There is no membrane
separating the nucleolus from the rest of the nucleus.
• Though most nuclei have one nucleolus, the number ranges from
zero to several because of their transient structure.
• Nucleoli appear as dark, dense, irregular shaped areas of fibers
and granules in the cell's nucleus.
• Only plant and animal cells contain nucleoli.
• They are made of proteins and ribonucleic acid, or RNA, and contain
proteins, ribosomal RNA, and ribosomes that are being synthesized.
• The genetic information needed to create ribosomal proteins is
copied from the nucleus by messenger RNA, which then takes it to
the cell cytoplasm.
• The cytoplasm then takes the information and transfers it to the
ribosomes in the cytoplasm.
• This protein then goes into the nucleus and with the other ribosmal
RNA subunits creates the two blocks that make up a complete
ribosome.
Transfer RNA (tRNA)
Aminoacyl-tRNA
Synthatase
An enzyme that adds amino
acids to the 3’ end of the free
tRNA
Translation Initiation
E, P and A sites
Each ribosome has a binding site for mRNA and three binding sites for tRNA
molecules.
The P site holds the tRNA carrying the growing polypeptide chain.
The A site carries the tRNA with the next amino acid.
Discharged tRNAs leave the ribosome at the E site
Polypeptide
Elongation
Polypeptide Elongation, cont’d.
Termination
When the stop codon enters the ribosome, a protein called a release factor
enters the A site and hydrolyzes the bond between the tRNA in the P site and
the last amino acid of the polypeptide chain. This frees the polypeptide and
breaks up the ribosomal subunits.
Elongation
• mRNA are translated in the 5’ – 3’
direction
• The polypeptide chain grows from the NH2
(N-Terminus) end to COOH (CTerminus)end
The Dictionary of The Genetic Code
UTRs
• The mRNA also contains regions that are not
translated. In eukaryotes the 5' untranslated
region and the 3' untranslated region contain a
methyl guanine cap and polyA tail respectively.
Polyribosomes
• Each mRNA is translated by multiple ribosomes.
• So each mRNA transcripts gives rise to multiple
polypeptide chains
• Polyribosomes Video
Polyribosomes in Prokaryotes
• Prokaryotes can
have transcription
and translation
occurring
simultaneously.
• WHY?
Get Help
• http://www.phschool.com/science/biology_
place/biocoach/translation/gencode.html
Bound versus Free Ribosomes and
the role of the Endoplasmic Reticulum
Some polypeptides have an ER signal
sequence. The signal sequence is
recognized by a Recognition Particle,
or SRP. This is then bound to a
receptor. This complex guides the
protein through a channel like
region. It also consists of a docking
site for the ribosome.
Those Funny Bases in tRNA
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Inosine (a form of Adenine, a pre-cursor to adenine) It is usually found in the
anticodon region
Pseudoruidine (most common modified base it is an isomer of uridine)
Dihydrouridine – is a uridine with 2 extra hydrogens – a more saturated version
of uridine
Thymine – although not usually found in RNA, it is in tRNA
Mutations
• Single-base Substitutions
• Single base Insertions and Deletions –
frame-shift mutations
Substitutions - Missense mutations
A single base, say an A, becomes replaced by another. Single base substitutions
are also called point mutations.
EXAMPLE: sickle-cell disease The replacement of A by T at the 17th nucleotide
of the gene for the beta chain of hemoglobin changes the codon GAG (for
glutamic acid) to GTG (which encodes valine). Thus the 6th amino acid in the
chain becomes valine instead of glutamic acid.
Substitutions – Nonsense Mutations
With a nonsense mutation, the new nucleotide changes a codon that
specified an amino acid to one of the STOP codons (TAA, TAG, or TGA).
Therefore, translation of the messenger RNA transcribed from this
mutant gene will stop prematurely. The earlier in the gene that this
occurs, the more truncated the protein product and the more likely that
it will be unable to function.
Substitutions - Silent mutations
Most amino acids are encoded by several different
codons. For example, if the third base in the
TCT codon for serine is changed to any one of
the other three bases, serine will still be
encoded. Such mutations are said to be silent
because they cause no change in their product
and cannot be detected without sequencing the
gene (or its mRNA).
Insertions and Deletions
Extra base pairs may be added (insertions) or removed (deletions) from the DNA
of a gene. This usually causes a shift in the reading frame and changes the amino
acid sequence of the protein from that point onward.
Frameshifts often create new STOP codons and thus generate nonsense
mutations. Perhaps that is just as well as the protein would probably be too
garbled anyway to be useful to the cell.
Each mRNA has 3 Reading Frames
• Each reading frame will translate
into a completely different
protein.
• However the correct reading
frame is set by several initiation
factors and by the fact that the
start codon for protein synthesis
is always AUG, which codes for
methionine.
• Therefore in all proteins, at least
as they are synthesized on the
ribosome, their first amino acid is
always methionine
Mutations: Thymine Dimers
• A thymine dimer is the covalent bonding of two
adjacent thymine residues within a DNA
molecule.
• Thymine dimers usually occur in response to
ultraviolet radiation or chemical mutagenic
agents.
• Create kinks in DNA
• Can be repaired by certain enzymes
Thymine Dimers
Xeroderma Pigmentosa
•Xeroderma pigmentosa, or XP, is an autosomal recessive genetic
disorder .
•In XP, thymine dimers in DNA caused by ultraviolet (UV) light cannot
be repaired due to mutations in the genes that code for the repair
enzymes. Therefore, the repair enzymes(proteins) are not made.
• This disorder leads to multiple basaliomas and other skin
malignancies at a young age.
•In severe cases, it is necessary to avoid sunlight completely.
•The two most common causes of death for XP victims are metastatic
malignant melanoma and squamous cell carcinoma.
•For some reason, XP is about six times more common in Japanese
people than in other groups.
Wobble Base Pairing
• The presence of more than one codon for a given amino acid
raises the issue of how many tRNAs are necessary to read
the code.
• You could have one for each codon, but it turns out that there
are less than this (~45).
• The reason has to do with what Crick called "wobble base
pairing".
• Wobble refers to non Watson-Crick base pairing that can take
place at the third position of the codon and first position of the
anticodon.
• The wobble pair is so-called because the base has shifted
("wobbled") in order to make the hydrogen bonding work.
The Wobble Rules
Wobble Rules: Read as 5' position in anticodon pairs with 3' position in codon:
G pairs with C or U;
C pairs with G;
A pairs with U;
U pairs with A or G;
I pairs with A, U, or C
Note that the anticodon position in the tRNA can also have the base
inosine (I), a purine that is not present in the messenger RNA (codon)
Euchromatin
• Euchromatin is DNA (true chromatin) that
is actively transcribed, because it contains
all the normally functional genes
• These regions of a chromosome are very
“relaxed” or least condensed during
interphase
• These parts of the chromosome stain
poorly or not at all;
Heterochromatin
• Heterochromatin on the other hand, is
densely staining and condensed
chromosomal regions
• It is, for the most part, genetically inert.
chromatin that remains tightly coiled (and
darkly staining) throughout the cell cycle.
Euchromatin vs. Heterochromatin
Role of histones
• They are positively
charged and
therefore, the
negatively charged
DNA molecule is
attracted to them
• They condense 6
feet of DNA so that
it would fit inside
the nucleus of
each cell
Histone Acetylation
• Acetylation of the lysine residues at the N terminus of
histone proteins removes positive charges, thereby
reducing the affinity between histones and DNA. This
makes RNA polymerase and transcription factors easier
to access the promoter region. Therefore, in most
cases, histone acetylation enhances transcription while
histone deacetylation represses transcription
• Histone acetylation is catalyzed by histone
acetyltransferases (HATs) and histone deacetylation is
catalyzed by histone deacetylases
DNA Methylation (CpG sites)
• DNA methylation may impact the transcription of genes
in two ways.
– First, the methylation of DNA may itself physically impede the
binding of transcriptional proteins to the gene, thus blocking
transcription.
– Second, and likely more important, methylated DNA may be
bound by proteins known as Methyl-CpG-binding domain
proteins (MBDs). MBD proteins then recruit additional proteins to
the locus, such as histone deacetylases and other chromatin
remodelling proteins that can modify histones, thereby forming
compact, inactive chromatin termed silent chromatin.
• Compared to the Xa, the Xi has high levels of DNA
methylation, low levels of histone acetylation
DNA Methylation
• The association between cytosine methylation and
transcriptional silencing in mammalian cells has become
well established, and a number of proteins that catalyze
the transfer of a methyl group to the 5-carbon of the
cytosine pyrimidine ring have been cloned and
characterized.
• These DNA methyltransferases (m5C-MTases) are
encoded by a diverse family of genes found in
prokaryotes as well as all four groups of eukaryotes.
• In mammals, cytosines are methylated predominantly in
the context of the 5'-CpG-3'(CG) dinucleotide, and the
majority of these sites are methylated. Only the short
CG-rich regions known as CpG islands are methylation
free in normal tissues.
Inactive (i) vs. active(a) X
• DNA packaged in heterochromatin, such
as the Xi, is more condensed than DNA
packaged in euchromatin, such as the Xa.
• The inactive X forms a discrete body
within the nucleus called a Barr body.
• The Barr body is generally located on the
periphery of the nucleus, is late replicating
within the cell cycle
Polytene Chromosomes
• Polytene chromosomes form when multiple rounds of replication
produce many homologous chromatids that remain synapsed
together.
• In addition to increasing the volume of the cell's nuclei and causing
cell expansion, polytene cells may also have a metabolic advantage
as multiple copies of genes permits a high level of gene expression.
• In Drosophila melanogaster, for example, the chromosomes of the
larval salivary glands undergo many rounds of endoreplication, to
produce large amounts of glue before pupation.
• Basically: Polytene chromosomes make nuclei larger and can
help the cell produce huge amounts of a particular protein.
Polytene chomosome from the salivary
gland of Drosophila melanogaster (stained)
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