Translation - protein metabolism

Question 1

DNA replication vs translation

Answer 1

• For translation and protein production – only certain DNA sections are transcribed into RNA and translated into protein
• For DNA replication all DNA information has to be copied into DNA

Question 2

rough ER

Answer 2

ribosomes attached to the outer (cytosolic) face of the endoplasmic reticulum (ER). The ribosomes are the numerous small dots bordering the parallel layers of membranes.

Question 3

adapter (tRNA) brings AA to mRNA

Answer 3

amino acid is covalently bound at the 3’ end of a tRNA molecule and that a specific nucleotide triplet elsewhere in the tRNA interacts with a particular triplet codon in mRNA through hydrogen bonding of complementary bases

Question 4

non-overlapping code

Answer 4

Overlapping versus nonoverlapping genetic codes. In a nonoverlapping code, codons (numbered consecutively) do not share nucleotides. In an overlapping code, some nucleotides in the mRNA are shared by different codons. In a triplet code with maximum overlap, many nucleotides, such as the third nucleotide from the left (A), are shared by three codons. Note that in an overlapping code, the triplet sequence of the first codon limits the possible sequences for the second codon. A nonoverlapping code provides much more flexibility in the triplet sequence of neighboring codons and therefore in the possible amino acid sequences designated by the code. The genetic code used in all living systems is now known to be nonoverlapping.

Question 5

multiple reading frames in an mRNA sequence

Answer 5

• Non-overlapping triplet genetic code without divisions between codons could be translated in three different reading frames – only the frame initiated by the most 5’ AUG start codon is the correct frame for most proteins.
• Frameshifting caused by base addition or deletion mutation changes the polypeptide sequence.
• Regions of sequence that are translated in more than one of the three possible reading frames are rare but known in prokaryotes and eukaryotes, and especially in their viruses.

Question 6

reading frame translation accuracy

Answer 6

• Each gene ensures that the correct reading frame is translated by having a universal start codon: AUG = initiation codon
• The tRNA with the correct anticodon to AUG carries the amino acid Methionine (Met)
• Met is often later removed in modification of the final protein.

Question 7

genetic code

Answer 7

• The code is written in the 5’
  –> 3’ direction.
• Third base is less important in binding to tRNA.
• AUG = initiation codon (->Met)
• The first codon establishes the reading frame.
  o If reading frame is thrown off by a base or two, all subsequent codons are out of order
• 61/64 codons code for amino acids.
• There are three termination codons:
  o UAA, UGA, UAG (STOP)
• Genetic code = dictionary
• Set of rules by which nucleotide information encoded in DNA template strand is translated into proteins
• mRNA sequence displayed in 5’-> 3’ direction = codon

Question 8

redundancy of genetic code

Answer 8

• Most amino acids have more than one codon
• Some codons are less subject to causing a mutation in an amino acid sequence because of degeneracy or because of the abundance of such tRNAs.
• There are 20 amino acids with 61 possible codons.
• Only Met and Trp have a single codon.

Question 9

universality of genetic code

Answer 9

• Same coding is used for different organisms e.g. by prokaryotes and eukaryotes, across species.
• Main exception are mitochondria:
  o Mitochondria contain DNA and use a slightly different code.: In vertebrate mtDNA UGA encodes Tryptophan (Trp) instead of a STOP codon, In vertebrate mtDNA AGA/AGG encodes for STOP codons instead of Arginine (Arg).
  o Mitochondria encode their own tRNAs, using 22 instead of 32
  o Divergent evolution

Question 10

base substitutions

Answer 10

• Base substitutions = Point mutations = Single Nucleotide Polymorphisms
• Replacement of one nucleotide in the DNA by another If that altered DNA gets transcribed it can lead to the same or an alternate amino acid contributing to the final protein product
  A. Synonymous (silent) mutation
  B. Missense mutation (nonsynonymous)
  C. Nonsense mutation

Question 11

silent mutation (synonomous)

Answer 11

• One nucleotide is changed (usually in 3rd position)
• Here T -> C mutation in DNA leads to A -> G change in mRNA codon
• Leads to identical amino acid
• no change in polypeptide due to degenerative genetic code
• Protein function unaffected

Question 12

missense mutation (nonsynonomous)

Answer 12

• Leads to an alternate amino acid
• Here Lysine gets replaced with Glutamate
• After translation the finished protein will contain one different amino acid
• Can result in non-functional protein

Question 13

nonsense mutation

Answer 13

• Change leads to a STOP codon (UAG or UAA or UGA)
• Results in shortened protein/polypeptide
• Usually results in non-functional protein

Question 14

base insertion or deletion

Answer 14

• Insertion or deletion of 1 or 2 nucleotides or multiples thereof change the reading frame = Frame shift mutation
• Usually lead to different consecutive amino acids if not multiples of three
• Usually several amino acids are changed/incorrect
• Ultimately result often in a premature STOP codon -> truncated protein
• any insertion or deletion mutation can alter the reading frame

Question 15

molecular recognition of codons in mRNA by tRNA

Answer 15

• The mRNA codon sequence is complementary to the tRNA anticodon sequence.
• The codon in mRNA base pairs with the anticodon in tRNA via hydrogen bonding.
• The alignment of two RNA segments is antiparallel.
• The tRNA is shown in the traditional cloverleaf configuration

Question 16

Genetic code resistance to mutations

Answer 16

• Degenerate code allows certain mutations to still code for the same amino acid.
  o “silent” mutations―different nucleotide in DNA but same amino acid in protein
• Mutation in the first base of a codon usually produces a conservative substitution.
  o Example: GUU –> Val but AUU –> Leu

Question 17

five stages of protein synthesis

Answer 17

1. Activation of amino acids: tRNA aminoacylated
2. Initiation of translation: mRNA and aminoacylated tRNA bind to ribosome
3. Elongation: cycles of aminoacyl-tRNA binding and peptide bond formation…until a STOP codon is reached
4. Termination and ribosome recycling: mRNA and protein dissociate, ribosome recycled
5. Folding and post-translational processing: catalyzed by a variety of enzymes

Question 18

eukaryotic ribosomes

Answer 18

• Ribosome Is a Key Player in Protein Synthesis
• Made of rRNA and protein
  o rRNAs form the core
  o RNA does the catalysis of peptide bond formation
• Made of two subunits bound together (60S and 40S => 80s)
• mRNA running through them

Question 19

bacterial ribosomes

Answer 19

• Ribosome Is a Key Player in Protein Synthesis
• Make up ~25% of dry weight of bacteria
• ~65% rRNA, 35% protein
  o rRNA forms the core
  o RNA does the catalysis of peptide bond formation
• Made of two subunits bound together (30S and 50S => 70s) in bacteria, with mRNA running through them

Question 20

tRNA characteristics

Answer 20

• ssRNA of 73–93 nucleotides in both bacteria and eukaryotes
• Cloverleaf structure in 2-D
• “Twisted L” shape in 3-D
• Most have G at 5’-end; all have CAA at 3’-end
• Have modified bases
  o methylated bases
• Amino acid arm
  o has amino acid esterified via carboxyl group to the 2’-OH or 3’-OH of the A of the terminal CAA codon
• Anticodon arm
• D arm
  o contains dihydrouridine (D)
  o contributes to folding
• TψC arm
  o contains pseudouridine (ψ)―has bonding between base and ribose
  o helps in folding

Question 21

stage 1 protein synthesis: activation of AA

Answer 21

− creation of Aminoacyl intermediate
o Aminoacyl-tRNA synthetases = family of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs
o Aminoacyl-tRNA synthetases esterify 20 amino acids to corresponding tRNAs: Requires ENERGY, ATP –>creates aminoacyladenylate intermediate, Pyrophosphate (PPi) is cleaved, (ATP-> AMP)

Question 22

stage 2 protein synthesis: initiation

Answer 22

(prokaryotes)
- The first tRNA is unique.
- The first codon of any peptide is AUG (Met).
- All organisms have two tRNAs for Met.
o In bacteria, plus chloroplasts and mitochondria, initiation tRNA inserts a special tRNAfMet (N-formylmethionine)
o Internal Met is inserted with normal tRNAMet.
- Eukaryotic protein begins with Met, (not fMet), but a special tRNA is still used for peptide initiation.

(eukaryotes)
- More complex mechanism than prokaryotes (over 12 IFs)
1. Initiation factors (IFs) bind to the 40S subunit
2. IFs mediate the association of, GTP charged initiator tRNAmet to form a 43S pre-initiation complex,
3. Then mRNA (with the 5’ cap shown in red) binds to form a 48S complex
4. This scans the mRNA looking for the start codon
5. The final 80S initiation complex is formed as the 60S subunit associates & initiation factors are released

Question 23

stage 3 protein synthesis: elongation

Answer 23

(prokaryotic – very similar to eukaryotic)
1: Binding of the incoming aminoacyl-tRNA (requires energy)
o Incoming aminoacyl-tRNA first binds to Elongation Factor (EF-Tu) –GTP complex.
o This complex binds to the A site of the ribosome initiation complex.
o GTP is hydrolysed, EF-Tu-GDP leaves the ribosome.

2: Peptide bond forms
o There are now two amino acids bound to tRNAs positioned for joining.
o One is on the A site, the other on the P site.
o N-formylmethionyl group is transferred from its tRNA in the P site to the amino acid in the A site.
o The reaction is catalyzed by the 23S rRNA (ribozyme).
o “Uncharged” (de-acetylated) tRNAfMet is now in the P site.

3:
o Translocation of the ribosome
o The ribosome moves one codon toward the 3’-end of the mRNA
o uses energy from GTP hydrolysis: GTP is part of EF-G (translocase)
o leaves A site open for new aminoacyl-tRNA

Question 24

stage 4 protein synthesis: termination

Answer 24

• Signaled by a stop codon
• UAA, UAG, or UGA in the A site will trigger the action of termination factors (release factors) RF-1, RF-2, RF-3.
• These help to:
  o hydrolyze terminal peptide-tRNA bond
  o release peptide and tRNA from ribosome
  o cause subunits of ribosome to dissociate so that initiation can begin again

features of protein synthesis
- Large energy cost
- Can be rapid when accomplished on clusters of ribosomes called polysomes
- In bacteria, tightly coupled to transcription
o Translation can begin before transcription is finished.
o No nucleus

Question 25

stage 5 protein synthesis: post-translational modifications

Answer 25

• Some proteins require modification before the fully active conformation is achieved.
• Post-translational modifications include:
  o enzymatic removal of formyl group from first residue, or removal of Met and sometimes additional residues
  o acetylation of N-terminal residue
• Removal of signal sequences or other regions
• Attaching carbohydrates
• Removing sequence to activate an enzymes

Question 26

protein targeting and degradation

Answer 26

• Proteins move from site of synthesis to:
  o exit a cell
  o become part of the membrane
• enter a subcellular compartment
• Most have a signal sequence at or near N-terminus
  o 13−36 amino acid residues in length
• This takes place in eukaryotic cells, where subcellular organization aids in compartmentalizing metabolic pathways.

Question 27

directing peptides to the ER and beyond

Answer 27

• As peptide emerges from the ribosome, the signal sequence is bound by signal recognition particle (SRP).
• SRP/ribosome/RNA complex is delivered to the ER lumen.
  o Some modification takes place here (glycosylation, etc.).
• Transport vesicles then take proteins to Golgi apparatus.
  o Proteins are sorted here in ways poorly understood.
• Proteins enter the biosynthetic/secretory pathway.
• Proteins for mitochondria and chloroplast bind chaperone proteins in the cytosol and are delivered to receptors on the exterior of the organelle.

Question 28

protein degradation inevitable

Answer 28

• Half-lives of proteins range from seconds to days to even months.
  o Hemoglobin is long lived.
  o Defective proteins are short lived, as are many metabolism regulatory proteins that respond to rapidly changing needs.
• But all are eventually degraded.
• In eukaryotic cells, two major pathways mediate protein degradation
  o Ubiquitin-proteasome pathway
  o Lysosomal proteolysis