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Who was the first to suggest a relationship between genes and the production of proteins (enzymes)?
Archibald Garrod
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Garrod studied patients with what disease?
alkaptonuria
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What is the one gene-one enzyme theory?
a single gene controlled the synthesis of a single enzyme
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Who proposed the one gene-one enzyme theory? What organism did they work with?
- George Beadle and Edward Tatum
- mutated fungi
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How did following researchers modify the one gene-one enzyme theory?
- enzymes are only one category of cellular proteins encoded by a gene
- some proteins are composed of two or more polypeptides
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Why is it more accurate to say that a structural gene encodes a polypeptide not necessarily a protein?
polypeptide denotes structure and protein denotes function
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What is a codon?
groups of three nucleotides on mRNA that codes (mostly) for a particular amino acid
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What is an anticodon?
found on tRNA and recognizes/corresponds to codons on mRNA
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What is the start codon and what does it code for?
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What are the stop codons?
- UAA
- UAG
- UGA
- *do not code for an amino acid
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Why three nucleotides per codon? Why not two?
if it only coded for 2, it could only code for 16 amino acids and there are 20. having 3 gives you multiple ways to code for an amino acid
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How is the genetic code degenerate?
because the number of possible codon combinations (64) exceeds the number of possible amino acids (20)
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What is a synonymous codon?
two codons that specifiy for the same amino acid
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What are codon families comprised of?
synonymous codons
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What position base is the wobble codon?
third
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Researchers have found the genetic code to be almost universal. What type of DNA is slightly different?
mtDNA in mammals
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What is the reading frame?
- the start codon (AUG) defines the reading frame of any mRNA
- indels shift the reading frame and it alters the amino acids creating a different polypeptide
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What is the N-terminus and the C-terminus?
- amino terminus end
- carboxyl terminus end
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What is the primary protein structure
the sequence of a chain of amino acids
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What is the secondary protein structure?
- occurs when a sequence of amino acids are linked by hydrogen bonds
- alpha helix and beta pleated sheets
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What is the tertiary protein structure?
occurs when certian attractions are present between alpha helices and pleated sheets
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What is the quartinary protein structure?
a protein consisting of more than one amino acid chain
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How are tRNA molecules named?
- according to the amino acid they carry
- i.e. tRNAphe
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What are some common features of tRNA?
- 3 stem loop structure
- a few vairiable sites
- acceptor stem with 3' single stranded region CCA sequence
- anticodon in 2nd stem loop
- ~75 nucleotides long
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If the anticodon in the tRNA is 5'-CGG-3', what would the complementary codon be?
5'-CCG-3'
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What is a charging tRNA?
- amino acid and ATP bound to enzyme; AMP binds to amino acid and pyrophosphate (PPi) is relseased
- activated aminio acid is attachd to the 3' end of tRNA at acceptor stem and AMP is released from enzyme
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What is the error frequency of a charging tRNA?
1 in100,000
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What is the wobble hypothesis?
- first 2 positions of codon adhere strictly to the AT/CG rule
- 3rd position can tolerate certain mismatches
- 1st position of the anticodon can move slightly so that hydrogen bonding can occur between mismatched codon and andticodon
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What are isoacceptors?
- two or more tRNAs that are able to recognize the same codon
- i.e. anticodon (CCA or CCG) both recognizes single codon GGU
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What gives additional flexibily to the tRNA molecule?
- the structure of bases in tRNAs (especially wobble position) can be modifed into less common nucleosides
- wobble interactions between anitocodon and codons
- G pairs with C or U
- C pairs with G
- A pairs with A or G
- U pairs with A or G
- I (inosine) pairs with A, U, or C
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Describe bacterial ribosome.
- sinlge type
- found in cytoplasm
- 1/3 cell mass
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Describe eukaryotic robosomes.
- biochemically distinct ribosomes
- most abundant type functions in the cytosol
- unique, smaller ribosomes are found in teh mitochondria and chloroplasts
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What is the structure of rRNA?
- composed of a large and small subunit
- overall shape result of rRNA since it constitutes most of the mass
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What is the interface between the small and large subunits primarily composed of?
rRNA
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What clusters on the outer suface of the ribosome and on the periphery of the interface?
proteins
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Bacterial ribosome= ___S
Small subunit= __S with ___ different proteins and ___S rRNA
Large subunit= ___S with ___ different proteins and two rRNa (___S + ___S)
- 70S
- 30S, 21, 16S
- 50S, 34, 23S, 5S
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Eukaryotic ribosome= ___S
Small subunit= ___S with ___ differnt proteins and __S rRNA
Large subunit= ___S with ____ different proteins and ___S + ___S rRNA
- 80S
- 40S, 33, 18S
- 60S, 49, 5S and 5.8S
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What are the tRNA binding sites?
- peptide site (P site)
- aminoacyl site (A site)
- exit site (E site)
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Overview of initiation.
mRNA, initiator tRNA (bound to start codon), and ribosomal subunits assemble
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Overview of elongation.
ribosome slides along mRNA in 5' to 3' direction moving over the codons sequentially linking amino acids brought in by tRNA
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Overview of terminaion.
stop codon signals the termination of translation at which time the polypeptide is released and ribosomal complex disassembles
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Describe the initiation stage in bacteria.
- the initiator tRNA is designated tRNAfmet because the methioinine has been modified
- a formyl group (__CHO) is attached to the nitrogen atom in methionine after it has been attached to the tRNA
- Shine-Dalgarno sequence promotes hyrdogen bonding of mRNA to the 30S subunit
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What is the Shine-Delgarno sequence?
a sequence that is complementary to a short sequence withing the 16S rRNA
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What three factors are required for mRNA, tRNAfmet, and ribosomal subunits to associate into initiation?
- IF1 (initiation factor)
- IF2
- IF3
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What does IF1, IF2, and IF3 do?
- IF1- separates and prevens premature binding of 30S and 50S subunits
- IF2- binds fmet-tRNA to 30S and binds to GTP
- IF3- binds 30S to mRNA
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Describe initiation in eukaryotes.
- additional intitation factors are required
- eIF designation for eukaryotes
- initiator tRNA carries methionine instead of formylmethionine
- eIF binds directly to tRNAmet prior to recruitment of 40S subunit
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How are eukaryotic mRNAs recognized by the ribosome since there is no Shine-Delgarno sequence?
- Cap-Binding protein I (CBPI) binds to 7-methylgranosine cap on mRNA and recruits other initiation factors
- these initation factors unwind any secondary structures in mRNA and promote binding to ribosome
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Where to start translation?
- after binding, ribosome slides along mRNA downstram of 5' cap searching for AUG codon
- it will not always start at the first AUG codon, it depends what is around it
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What is Kozak's rule?
- consensus sequence for optimal translation initiation
- three places upstream tends to be a G and right next to the start codon is a g
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When does the elongation process begin?
when the start codon is identified and the 60S subunit assembles with the aid of eIF5
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Describe the elongation process.
- amino acid added one at a time to the growing polypeptide chain
- 15-18 amino acids per second in prokaryotes
- 6 amino acids per second in eukaryotes
- GTP hydrolysis is required
- 3 elongation factors involved
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When does termination occur?
when stop codon (nonsense codon) is reached in mRNA
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What is the stop codon recognized by?
proteins known as release factors which mimic the structure of tRNA
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How many release factors are needed to recognize the three stop codons in bacteria? In eukaryotes?
- multiple RF
- a single release factor (eRF)
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Describe the final step in termination.
- disassembly if mRNA, ribosomal subunits, and the release factor
- RF binds to site A
- bond between polypeptide and tRNA is hyrodylzed releasing it from the ribosome
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What are polyribosomes?
- mRNA transcript tht has many bound ribosomes in the act of translation
- in bacteria, translation can occur before transcription is finsihed
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Kanamycin
Streptomycin
Tetracylcine
Kirromycin
Puromycin
Erythomycin
- interferes with correct wobble base pairing
- prevent correct pairing of codon and anticodon
- prevent aminoacyl-tRNA binding at A site
- prevents EF-Tu disassociation from ribosome and process stalls in initiation phase
- premature chain termination
- blocks E-site
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What are sorting signals?
signals that direct amino acids into correct location
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Why is the process more complex in eukaryotes?
because of the compartmentalization
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What is cotranslational sorting? posttranslational sorting?
- sorting that can occur during tranlation
- sorting that can occur after translation is complete
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