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Molecular Biology
DNA Expression
CHAPTER 9
PART B
DNA Expression: The Central Dogma
oGenes contain specific sequences of bases coding the instructions for
proteins
◦ In general one gene codes for one protein
oThe basic process starts in the nucleus where enzymes transcribe the gene
to make a strand of RNA. The RNA exits the nucleus through the nuclear
pores. In the cytoplasm the RNA is translated into a sequence of amino acids
(the building blocks of proteins)
DNA
RNA
Transcription
(Nucleus)
Protein
Translation
(Cytoplasm)
Transcription
mRNA
processing
Assembly of RNA on unwound regions of DNA
molecule
mRNA
rRNA
tRNA
proteins
mature mRNA
transcripts
Translation
At an intact
ribosome,
synthesis of
a polypeptide
chain at the
binding sites
for mRNA
and tRNAs
ribosomal
subunits
Convergence
of RNAs
mature
tRNA
cytoplasmic
pools of
amino acids,
ribosomal
subunits,
and tRNAs
Final protein
Transcription: from DNA to mRNA
oTranscription is the first step in gene expression
oTranscription occurs in the nucleus in eukaryotes
oThe base sequence of a gene in DNA is copied to make a
single strand of RNA
Transcription: from DNA to mRNA
oDNA is unwound
oOnly the part of the chromosome containing the gene is unwound
oMakes a transcription bubble
Transcription: from DNA to mRNA
oRNA polymerase attaches to a binding site called a
promoter
oThe promoter is just in front of the gene (“up stream”)
Transcription: from DNA to mRNA
oRNA polymerase adds nucleotides one at a time using the
DNA sequence as a template
◦ A-U, T-A, G-C, C-G
Transcription: from DNA to mRNA
oTranscription ends when the polymerase passes the end of
the gene
Eukaryotic mRNA Processing
oOnce RNA is transcribed it will
be modified before leaving the
nucleus for the cytoplasm
◦ Introns are spliced out
◦ Eukaryotic genes contain
◦ Exons which are coding regions
◦ Introns which are non-coding regions
◦ Before the RNA leaves the nucleus the
introns are snipped out leaving just the
exons
Eukaryotic mRNA Processing
oOnce RNA is transcribed it will
be modified before leaving the
nucleus for the cytoplasm
◦ Protection from cytoplasmic RNA enzymes
◦ Poly A tail
◦ 50-300 adenines are added to the “tail” end (3’ end)
◦ 5’ “cap”
◦ A modified guanine is added to the “front” end (5’
end)
Translation
oTranslation is the second step in gene expression
oTranslation occurs in the cytoplasm
oThe base sequence in the mRNA is decoded into a
sequence of amino acids in a protein
Translation
oThe Genetic Code
◦ The sequence of nucleotides/bases in the mRNA “spell” out the
instructions for what protein the cell should make
◦ Different proteins determine the traits of cells and organisms
◦ Each set of three bases is one “word” or codon
◦ A codon indicates which amino acid to add to the protein
◦ Amino acids are protein monomers/subunits
Translation
oThe Genetic Code
◦ There are 64 different codons
◦ 61 codons code for 20 amino acids
◦ Thus most amino acids are encoded by more
than one codon
◦ 1 codon is used as a start signal
◦ AUG (codes for methionine)
◦ 3 codons are used as a stop signal
◦ UAA, UGA, UAG
Translation
oThe Genetic Code
5’
ATG TCG GAC CTA 3’
3’ TAC AGC CTG GAT 5’
5’
AUG UCG GAC CUA 3’ (mRNA)
met ser asp leu
(start)
Translation
otRNA
◦ Each tRNA has two attachment sites
◦ One is an anticodon
◦ A triplet of bases complimentary to mRNA codons
◦ The other attaches to the amino acid specified
by the codon
Translation
oInitiation
◦ Translation is initiated when the initiator tRNA
binds the first AUG (start codon) of the mRNA
and a small ribosomal subunit, then a large
ribosomal subunit joins them
◦ Initiator tRNA’s anticodon base pairs with AUG
◦ Amino acid is methionine (met)
◦ Initiator tRNA + mRNA + small and large
ribosomal subunits = initiation complex
Translation
oElongation
1. Initiator tRNA+met lines up in the P site of the ribosome
◦
◦
Anti-codon matches AUG
Carries methionine
2. Second tRNA+aa2 is placed in the A site of the ribosome
◦
The second tRNA’s anticodon matches the next three bases
(codon) on the mRNA
Carries the appropriate amino acid
◦
◦
called aa2 to indicate it is the second amino acid
3. Peptide bond forms between met and aa2
4. Initiator tRNA is released from the P site and from its
amino acid, met
Translation
oElongation
5. The second tRNA+aa2-met complex moves to the P site
of the ribosome
6. Third tRNA+aa3 moves into the now vacant A site of the
ribosome
7. Peptide bond forms between aa2 and aa3
◦
Forms a polypeptide consisting of three amino acids
◦
Met-aa2-aa3
8. This process continues adding an amino acid for each
codon
9. The polypeptide elongates
Translation
oTermination
◦ The ribosome encounters a stop codon in the mRNA
◦ There are no matching tRNAs for stop codons
◦ A release factor binds
◦ All the parts of the complex dissociate
◦ mRNA (can be used again)
◦ Small and large ribosomal subunits (can be used again)
◦ New polypeptide chain
◦ The new polypeptide will either stay in the cytoplasm or enter the rough endoplasmic
reticulum of the endomembrane system
Mutations
oMutations are changes in the nucleotide sequence of DNA
oThese can affect the sequence of amino acids in the
encoded polypeptide thus affecting the function of the
protein
Mutations
oCommon gene mutations
◦ Base-pair substitution
◦ A single base pair changes
◦ Affects one codon and thus one amino acid
Mutations
oCommon gene mutations
◦ Insertions
◦ One or more base pairs are added to the original DNA sequence
◦ Results in a reading frame shift
Mutations
oCommon gene mutations
◦ Deletions
◦ One or more base pairs are removed from the original DNA sequence
◦ Results in a reading frame shift
Mutations
oCommon gene mutations
◦ Frameshift (due to insertion or deletion)
◦ Shifts the 3-bases-at-a-time reading frame
◦ Changes the amino acid sequence from the point of mutation on.
◦ The cat ate the rat
◦ Tec ata tet her at (deletion)
◦ Thr eca tat eth era t (insertion)
Mutations
oMutation example: Sickle-cell anemia
◦ In the DNA sequence coding for the beta chain in hemoglobin there is a
single base-pair substitution
◦ Hemoglobin carries oxygen through the blood
◦ When that DNA is transcribed to mRNA the mistake is copied
◦ When the mRNA is translated into a polypeptide one different amino
acid is used
◦ Normal sequence:
◦ Mutated sequence:
val-his-leu-thr-pro-glu-gluval-his-leu-thr-pro-val-glu-
◦ This one change disrupts the normal 3D folding of the protein
◦ Mis-shaped hemoglobin causes red blood cells to distort into a sickle shape under
low oxygen conditions
◦ Causes clotting and disrupts blood circulation
Summary
oDNA expression
◦ Genes “code” for proteins and thus traits
oTranscription
oTranslation
◦ Genetic code
oGene Mutations