notes lecture 11 bi 102 transcription and translation · _ y z z x y a ^ ' v ] > ] v p µ ]...
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BI 102
Lecture 11:Transcription and Translation
Genetic Code
• There are 4 nucleotide bases (A, G, C, T)• The sequence of nucleotide bases in DNA is a code for making
proteins• What’s the primary business of any cell?
• Proteins are built from amino acids• There are 20 amino acids• How do 4 nucleotide bases code for all 20 amino acids?
Genetic Code
• Combinations of 3 nucleotide bases form 64 unique codes• These combinations code for the 20 amino acids
• Some redundancy
Number of bases Total number of unique codes
1 base forms code pattern 41 = 4 unique codes (A, G, C, T)
2 bases form code pattern 42 = 16 unique codes
3 bases form code pattern 43 = 64 unique codes
4 bases form code pattern 44 = 256 unique codes
Genetic Code
• A group of 3 DNA bases that code for a single amino acid is called a triplet
Genetic Code
• DNA is too large, too precious to leave the nucleus• Protein-making machinery is in the cytosol• How does DNA direct protein synthesis from within the nucleus?
Transcription
• Solution: the cell makes a temporary copy in the form of mRNA• Process is called transcription
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Transcription
• Steps in transcription1) Initiation2) Elongation3) Termination
Transcription
• Initiation• Chromatin unwinds to expose DNA of the gene to be transcribed• DNA “unzips”• Enzyme concept called RNA polymerase bind to the promoter region of the
gene of interest
Transcription
• Elongation• One DNA strand is used as a template
• Called the template or antisense strand• Other strand is called the coding or sense strand
• mRNA nucleotides are added to match the DNA bases on the template strand
Warning: many, MANY online resources have these strands labeled backwards
Transcription
• Termination• Enzymes and mRNA strand are released• DNA “rezips”
Transcription
• DNA triplets are recoded as mRNA codons• Contains the same nucleotide
bases as the DNA sense strand • Except T in DNA is replaced with
U in mRNA
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mRNA Processing
• mRNA is not yet ready to direct protein synthesis• Three modifications must take place
1) 5’ capping2) 3’ polyadenylation3) mRNA splicing
mRNA Processing
• Enzymes in the cytosol rapidly degrade mRNA• Major defense against viruses
• Both ends of the mRNA molecule must be “capped” to protect it from these enzymes
mRNA Processing
• 5’ capping• Adds a G nucleotide by a unique type of bond• Prevents enzymes from breaking the bond and degrading the mRNA• Also marks which end of the mRNA represents the beginning of the protein
• Prevents the ribosomes from reading the strand backwards
mRNA Processing
• 3’ polyadenylation• Other end of the mRNA strand is trimmed of its final few nucleotides• Then capped with a tail of between 100 and 250 adenine nucleotides• Called the poly-A tail
mRNA Processing
• mRNA splicing• mRNA contains non-coding regions called introns• Protein-coding sections of mRNA are called exons• Introns are interspersed between the exons and must be removed
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mRNA Processing
• mRNA splicing• Introns are spliced out and degraded• Exons are joined together to create the final transcript
mRNA Processing
• Purposes of introns• Protects transcript
• Reduces chances that mutations will occur in coding regions of the transcript
• Allows multiple versions of a protein to be coded for by the same region ofDNA
• Exons can be spliced together differently to create multiple versions of the same protein
• Called splice isoforms or splice variants
Protein Synthesis
• Once a strand of mRNA has been processed, it is ready to be decoded to build a protein
• Process is called translation
• The genetic code is translated from the language of nucleic acids tothe language of proteins
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Genetic Linguistics
• Think about different written languages• Do the same letter combinations mean the same thing in all languages
that use the same alphabet?
English French
OursPain
Pet
Genetic Linguistics
• What about the languages of DNA and proteins?• All cells use the same nucleotides and the same amino acids
• But will every species read DNA in the same way?• If we put a gene for a protein from one species into another, will it
make the same protein?
Genetic Linguistics
• Experiment• Some jellyfish glow green under UV light• Due to the presence of a protein called green fluorescent protein
(GFP)
Genetic Linguistics
• Experiment• What will happen if we take the gene that codes for GFP and put it
into bacteria?• Will the bacteria make GFP?• Or will they translate the genetic code differently?
+ = ?
Genetic Linguistics
• Experiment• They make GFP!
+ =
Genetic Linguistics
• What about other species?• How will they translate jellyfish DNA?
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Genetic Linguistics
• They make GFP too!• What does this tell you about the genetic code?
Genetic Linguistics
• They make GFP too!• What does this tell you about the genetic code?• It’s universal!• An mRNA codon will be translated into the same amino
acid, no matter the species• We exploit this in so many, very cool ways
• Recombinant DNA technology
Translation
• How do we get from mRNA to protein?• Takes place on the ribosome• Involves all 3 types of RNA
• mRNA• rRNA• tRNA
Translation
• Ribosomes are constructed of rRNA and proteins• Composed of two subunits
• Small subunit• Large subunit
• The two subunits come together around an mRNA molecule
• Move along the mRNA strand as the transcript is read
Translation
• Take a look at transfer RNA (tRNA)• Note the anticodon loop at the bottom
• Group of 3 nucleotide bases that form complimentary base pairs with the codons of mRNA
Anticodon loop
Translation
• Take a look at transfer RNA (tRNA)• Note the amino acid attached to the top• A tRNA with a given anticodon always carries the same amino acid
Anticodon loop
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Translation
• Ribosome moves along mRNA to sequentially read each codon
• As successive mRNA codons are read, each is matched to itscorresponding tRNA anticodon
• Amino acid from the other end of tRNA is added to the growingprotein chain
Translation
• Amino acids are joined together to form a protein’s primary structure
• Takes about 20 seconds to form the whole protein
Translation
• The signal for where to begin translation is the mRNA codon AUG
• Called the START codon• Codes for the amino acid methionine• All proteins begin with methionine
• Translation ends when a STOP codon is reached• Codes for no amino acid• Has no corresponding tRNA• Ribosome comes apart and releases mRNA, protein
Translation
• Translation animation
Universal Genetic Code DNA Mutation and Repair
• DNA is precious• If the DNA code is changed, the mRNA transcript will change,
could change the protein that is made• Just one change in a nucleotide base can be devastating
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DNA Mutation and Repair
• Many factors lead to DNA changes• Happens constantly• Is usually repaired
DNA Mutation and Repair
• Many different DNA repair mechanisms exist• Type used depends on the type of damage• Correct sequence determined by referencing
• Complimentary strand• Homologous chromosome
DNA Mutation and Repair
• Example: mismatch repair in newly synthesized DNA
DNA Mutation and Repair
• Example: homologous end joining• Uses homologous chromosome
to fill in the gap
DNA Mutation and Repair
• Rarely, DNA damage is undetected or improperly repaired• Called a mutation
• Several types of mutations exist• Point mutation• Addition• Deletion• Frameshift• Inversion• Translocation
DNA Mutation and Repair
• Let’s start by decoding a correct mRNA sequence from a normal gene
AAA – ACG – UGG – CGC - AAG
Amino acid 1 Amino acid 2 Amino acid 3 Amino acid 4 Amino acid 5
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DNA Mutation and Repair
• Now let’s introduce a point mutation• Replace a single nucleotide with a different one
• Let’s replace the G at position 9 with a C
AAA – ACG – UGC – CGC - AAG
Amino acid 1 Amino acid 2 Amino acid 3 Amino acid 4 Amino acid 5
DNA Mutation and Repair
• This point mutation changed an amino acid• Lots of things could happen as a result…
DNA Mutation and Repair
• Nothing• Example: ACC to ACA• Both code for THR• No change in the protein
• Truncation• Example: UGG to UGA• UGG = TRP• UGA = STOP• Cuts the protein short
DNA Mutation and Repair
• Protein too long• Example: UGA to UGG• UGA = STOP• UGG = TRP• Stop codon isn’t made, so the protein is too long
• Protein isn’t made at all• Example: ATG to ATA• ATG = START• ATA = ILE• No START codon, so translation is never initiated
DNA Mutation and Repair
• Let’s go back to the original sequence and introduce an addition mutation
• Extra nucleotides are added
• Let’s add UU between positions 1 and 2
AUU – AAA – CGU – GGC – GCA - AG
Amino acid 1 Amino acid 2 Amino acid 3 Amino acid 4 Amino acid 5
DNA Mutation and Repair
• Let’s go back to the original sequence and introduce a deletion mutation
• Nucleotides are removed
• Let’s remove the C at position 5
AAA – AGU – GGC – GCA - AG
Amino acid 1 Amino acid 2 Amino acid 3 Amino acid 4 Amino acid 5
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DNA Mutation and Repair
• When a mutation changes how codons are grouped and read, it is called a frameshift mutation
• Changes every amino acid following the mutation
• Example: Take a sentence with all 3-letter wordsTHE DOG SAW THE CAT
• If we delete the E at position 3 and regroup the letters, it no longermakes sense
THD OGS AWT HEC AT• Which mutations in our previous examples produced a frameshift?
DNA Mutation and Repair
• Inversion and translocation• Large pieces of DNA (sometimes most of a chromosome) are
broken off and reattached elsewhere• Sometimes within the same chromosome• Sometimes to another chromosome
DNA Mutation and Repair
• May be benign• Entire genes and their promoters simply moved from one location
to another• Referred to as balanced• Frequently results in infertility, as gametes
are often unbalanced
DNA Mutation and Repair
• May result in significant impairment • Gene may be split into pieces, no longer code for a functional
protein• Example: most severe hemophilia is caused by an inversion of the
gene for a blood clotting protein
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