Lecture 8: The Central Dogma - From DNA to RNA to protein Part 2

Question 1

Transcription

Answer 1

• DNA —> RNA
• One-to-one correspondence of subunits
• Essentially the same language, except for minor changes (Thymine to Uracil, deoxyribose to ribose)

Question 2

Translation

Answer 2

• RNA —> protein
• No one-to-one correspondence: 20 amino acids, but only 4 bases
• Totally different chemical language

Question 3

Codon

Answer 3

• A set of 3 nucleotides

- Since there are more codons than amino acids, most amino acids have multiple corresponding codons

Question 4

Degenerate code

Answer 4

• Refers to the code corresponding between RNA sequence and amino acids
• You can’t be certain what the RNA sequence was for a specific protein since there are multiple codons for one amino acid

Question 5

Double nucleotide code

Answer 5

• 4 x 4 = 16 different combinations

Question 6

Triple nucleotide code

Answer 6

• 4 x 4 x 4 = 64 different combinations

Question 7

What does it mean for there to be multiple codons for one amino acid?

Answer 7

• It means that the amino acids with more codons are more common

Question 8

Transfer RNAs (tRNAs)

Answer 8

• Match amino acids with codons
• Short RNAs with distinctive 3D structure
• Contains a loop with an “anticodon” that is complementary to the appropriate amino acid’s codon

Question 9

Structure of tRNAs

Answer 9

• Resembles the shape of a clover leaf
• 3 stem loops
• In 3D structure, the D and T loops associate with each other to form a bent L-shaped structure

Question 10

Aminoacyl-tRNA synthetase

Answer 10

• Job is to attach an amino acid to its corresponding tRNA
• Has to recognize more than one tRNA because each amino acid could have multiple anticodons that correspond to it
• Distinct to each amino acid

Question 11

Attach amino acid to tRNA

Answer 11

• Amino acid is first “activated” by conjugation to AMP
• Energetically expensive, since both high-energy phosphates are used up in the process
• Amino acid is then transferred from AMP to tRNA
• Energy to make this bond comes from the “activation” of the amino acid in the previous step
• Resulting conjugate has high-energy bond between amino acid and tRNA
• Once complete, the synthetase proofreads for accuracy

Question 12

N-terminal —> C-terminal

Answer 12

• Direction in which protein synthesis occurs

- New amino acid is added to C-terminal end of growing chain

Question 13

Process of creating a polypeptide chain

Answer 13

• Peptide chain is attached to the last tRNA that was added
• The entire chain is added to each new amino acid added
• New aminoacyl tRNA replaced old tRNA, extending the chain by one residue

Question 14

Ribosome

Answer 14

• RNA message is decoded by this
• rRNAs make up structural and catalytic core: ribozyme
• Two subunits: large and small
• Makes sense why ribosome is made up of RNA instead of proteins in evolutionary perspective
• Reads a mRNA from 5’ —> 3’, reading 3 bases at a time
• Each mRNA has 3 potential “reading frames”, so it must choose one and remain consistent, or garbled translation occurs

Question 15

How does translation begin?

Answer 15

• With the codon AUG (Met) - this uses a special “initiator” tRNA, which is different from the Met tRNA used for the rest of translation

Question 16

Elongation

Answer 16

• Facilitated by elongation factors (EF-Tu/EF-G in prokaryotes, EF1/EF2 in eukaryotes), which use GTPase activity to allow proofreading and to speed up ribosome translocation
• There is also a 3rd proofreading step at the initiator complex to make sure that the correct initiator tRNA was brought in with Met
• 3 total proofreading steps in translation

Question 17

Termination

Answer 17

• When the ribosome encounters a “stop” codon (UAA,UAG,UGA)
• Instead of a tRNA, a release factor binds to the ribosome, causing the hydrolysis of the peptidyl tRNA, releasing the completed protein
• The ribosome then dissociates into separate small and large subunits, releasing the mRNA, release factor, and remaining tRNA
• Water is used to hydrolize the reaction of cleaving the polypeptide chain off of the last tRNA

Question 18

Polyribosomes

Answer 18

• A single mRNA may thus have several ribosomes translating simultaneously
• Each ribosome can only synthesize one peptide chain at a time, but a cell may read many copies of a protein for every copy of the relevant mRNA
• Cells don’t wait for one ribosome to finish before having the next one start - once the first ribosome has moved far enough along, another can bind to the cap

Question 19

Benefits of 5’ end of mRNA communicating with 3’ end

Answer 19

• One benefit of this is that the ribosome knows that the mRNA is intact if the 5’ and 3’ ends are in communication with each other
• Another benefit is that once the ribosome dissociates off of the 3’ end, it can quickly reattach to the 5’ end, which facilitates a continuous translation process

Question 20

Antibiotics and Protein Synthesis

Answer 20

• Many important antibodies are protein synthesis inhibitors
• Most affect only prokaryotes, due to differences between prokaryotic and eukaryotic ribosomes
• Sometimes also affect ribosomes in mitochondria and chloroplasts due to eukaryotic ribosomes within these organelles being similar to prokaryotic ribosomes
• A few exceptions can block eukaryotic ribosomes - useful in cell biology research
• Cycloheximide blocks translocation reaction (eukaryotic only)
• Puromycin mimics aminoacyl-tRNA and is incorporated into the growing polypeptide chain, causing premature termination (both eukaryotic and prokaryotic)
• Be aware that most common antibiotics are actually protein translation inhibitors

Question 21

RNA and protein synthesis are very energetically costly

Answer 21

• 10 ATPs per amino acid added at least
• Each elongation step requires multiple ATP/GTP molecules
• Adding each subunit requires the conversion of an NTP into an NMP, equivalent of 2 ATPs to ADPs
• mRNA splicing and protein proofreading use up even more ATP/GTP molecules

Question 22

Information of protein sequence

Answer 22

• Information for 3D-structure, cellular location, and protein function

Question 23

Information of mRNA

Answer 23

• Same information as protein plus information about initiating and terminating translation (and sometimes mRNA stability)

Question 24

Information of DNA

Answer 24

• Same information as mRNA plus information about initiating and terminating transcription, splicing (and anything useful in the introns themselves)