Proteome

Question 1

Ribosome small subunit

Answer 1

associated with the mRNA (35 bp of mRNA are bound during translation). mRNA located in the surface close to the junction between both subunits.

Question 2

Large subunit

Answer 2

catalyzes peptide bond formation
No contact with mRNA

Question 3

A-site

Answer 3

The next aminoacyl-tRNA loaded with a new amino acid enters through the A-site. This site exposes the mRNA codon representing the next amino acid, where the codon-anticodon interaction takes place.

Question 4

P-site

Answer 4

Contains the peptidyl-tRNA, a tRNA carrying the growing polypeptide chain. The polypeptide is transferred to the aa carried by the aminoacyl-tRNA in the A-site. Peptide bond catalyzed by large subunit.

Question 5

E-site

Answer 5

Deacylated tRNA (it has no amino acid nor polypeptide attached) exits the ribosome

Question 6

Initiation of translation requires

Answer 6

free ribosome subunits and initiation factors to assemble together forming the initiation complex at a specific position of the mRNA, the Ribosome binding site (RBS, Shine-Dalgarno sequence)

Question 7

The consensus RBS sequence for E. coli

Answer 7

5’-AGGAGGU-3’

Question 8

Initiator tRNA-fMet

Answer 8

used only for initiation of translation, it carries a formylated Met residue in the amino group, generating N-formyl-methionyltRNA
Two-stage reaction:
1) charge tRNA with Met by Aminoacyl-tRNA synthetase,
2) formylation reaction blocks amino group to prevent participation in chain elongation.

Question 9

Methionine.

Answer 9

Synthesis of all polypeptides starts with this aminoacid.
Three start codons:
AUG
GUG
UUG

Question 10

Two types of tRNAs carry Methionine:

Answer 10

Initiator tRNA-fMet,
Elongator tRNA-Met, used during elongation

Question 11

Initiation of translation in Bacteria: Steps

Answer 11

1. rRNA 16S of the small subunit recognizes the RBS. This allows the small subunit + IF-3 +IF-1 to bind to the mRNA over the initiation codon.
2. IF-2-GTP binds to the P-site and brings the initiator tRNA-fMet. IF-2 ensures that only tRNA-fMet starts translation.
3. IF-1 induces a conformational change in the initiation complex that enables attachment of the large subunit. It requires energy.
4. IF-2 has ribosome-dependent GTPase activity, it hydrolyzes a GTP molecule as energy source.
5. IFs and GDP are released
6. When the translation starts, the initiator tRNA-fMet is removed

Question 12

Initiation of translation in Eukaryotes

Answer 12

Initiation of translation in eukaryotes is similar to bacteria but more complex, with more IF. The main difference is how the small subunit finds the binding site for initiation (No RBS in eukaryotes). There are two types:
Cap-dependent initiation
Cap-independent initiation

Question 13

Cap-dependent initiation (steps)

Answer 13

1. A complex of initiation factors and initiator tRNA
   bind to the small ribosomal subunit (40S), forming
   the preinitiation complex.
2. A second group of initiation factors binds to the 5’
   methylated end of the mRNA to form the capbinding
   complex. It binds to the Poly-A binding protein (PABP) in the 3’ end, creating a circular structure (stimulates translation)
3. Preinitiation complex binds to the 5’ end of the mRNA and scans for the initiation codon
4. IFs dissociate and large subunit attaches

Question 14

Cap-independent initiation (viral RNAs)

Answer 14

The small ribosome subunit associates directly with an internal site of the mRNA of some viral RNAs, called internal ribosome entre site (IRES). There are different types of IRES and they have similar functions to bacterial RBS

Question 15

Transcript-specific regulation,

Answer 15

acts on a single transcript or small group of transcripts coding for related proteins

Question 16

Autoregulation of ribosome protein synthesis in E. coli

Answer 16

mRNA codes for two ribosomal proteins, L11 and L1. L1 is one of the largest rproteins and has a dual function (two binding sites):
1. If there are free rRNA (dissociated ribosomes) in the cytosol, L1 binds to the 23S rRNA -> It stimulates translation and helps building new ribosomes
2. If all rRNA are assembled into ribosomes  L1 binds to the RBS of its own mRNA -> Acts as a translational repressor blocking the initiation of translation
-> stops synthesis of ribosomes

Question 17

Secondary mRNA structure controls translation initiation

Answer 17

• It happens in viruses.
• mRNA secondary structure controls translation initiation so genes are translated in a set order
Initiation codons are bound together forming a loop structure in the mRNA. Only the first initiation site is accessible to the ribosome. During translation, ribosomes disrupt the secondarystructure and expose the second initiation site.

Question 18

Initiation is regulated by short non-coding RNAs

Answer 18

It happens in some bacteria
Short non-coding RNAs attach to recognition sequences within the mRNAs.
They can repress or stimulate initiation of translation
• Bind to the RBS of an mRNA blocks translation
• Bind to mRNA disrupts its secondary structure, making it accessible to ribosomes. Stimulates translation

Question 19

Elongation factor (EF-Tu)

Answer 19

Mediates the entry of a new aminoacyltRNA in the A-site. The process is similar in eukaryotes

Question 20

entry of the aa-tRNA

Answer 20

1.EF-Tu + GTP + aminoacyl-tRNA forms a ternary complex, and binds to the A-site of the ribosome.
2. First contact: Codon-anticodon interaction that stabilizes the tRNA binding
3. The 3’ end (CCA) of the aa-tRNA moves to the A-site of the large subunit (necessary for contact and peptide bond formation)
4. EF-Tu hydrolyzes GTP and Ef-Tu-GDP released. It is inactive and cannot bind aa-tRNA
5. Ef-Tu will be activated again with the EF-Ts and GTP

Question 21

Translocation of the ribosome:

Answer 21

1. Ribosome advances three nucleotides (a triplet) along the mRNA
2. The deacylated tRNA exits via E-site, the new peptidyl-tRNA is located in the P-site and a A-site is empty again and contains a new codon, so a new aminoacyl-tRNA can enter.
3. Elongation cycle is repeated until the STOP codon is reached

Question 22

Unusual elongation - Frameshifting

Answer 22

Ribosome may cause a frameshifting by skipping a base when it reads the mRNA, or by reading a base twice. Changes reading frame, leads to aberrant proteins. Spontaneous frameshifts occur randomly

Question 23

Programmed frameshifting

Answer 23

A single gene, dnaX, codes for two different subunits of the same protein (τ, γ), the DNA polymerase III of E. coli
• Full length translation produces subunit τ
• Programmed frameshift leads to a shortened subunit γ
Several factors stimulate the frameshift:
1) Hairpin loops stall the ribosome,
2) RBS-like sequence stalls ribosome binding 16S, and
3) weak codon-anticodon interactions (AAG)

Question 24

Termination of translation in Bacteria

Answer 24

Protein synthesis ends when the ribosome reaches one of
the 3 termination codons (UAA, UAG, UGA). Release
factors recognize the stop codon and stimulate the
termination of translation.

Question 25

Protein release factor (RF1)

Answer 25

has a structure that mimics tRNA, it enters in the A-site and releases the polypeptide.

Question 26

ribosome recycling factor (RRF)

Answer 26

has also a tRNAlike structure. Enters the A and P sites and dissociates the remaining components (tRNA, mRNA, large and small subunits)

Question 27

Proteome

Answer 27

Is the complete set of proteins synthesized in an organism/tissue/cell at a specific point in time. It changes over time. rRNA and tRNAs are produced when the cell is growing, to synthesize more proteins Cell grows -> it needs proteins -> synthesizes ribosomes and tRNA ->protein production increases If there is enough ATP, the cell divides and ribosomes are produced. Concentration of ATP in the cell regulates the production of rRNA and tRNA

Question 28

Stringent response

Answer 28

survival mechanism that shuts down synthesis of tRNA and ribosomes is activated in bacteria under nutrient limitation conditions. Bacteria reduces number of activities and focuses in the biosynthesis of amino acids.

Question 29

The stringent response: how it works

Answer 29

When a deacylated-tRNA enters the A-site, translation stops. Then the RelA protein (stringent factor) mediates the stringent response. RelA catalyzes the synthesis of ppGpp (guanosine tetraphosphate) and pppGpp (guanosine pentaphosphate), the alarmones. Alarmone activates the stringent response: • Inactivates the synthesis of ribosomes (tRNA and rRNA) • Activates the biosynthesis of amino acids

Question 30

protein folding

Answer 30

a physical spontaneous process by which a protein folds into its native 3D structure, becoming active. It is guided by hydrophobic interactions. All the information that the polypeptide needs to fold in the proper tertiary structure is in its amino acid sequence.

Question 31

Folding pathway

Answer 31

represented as a energy funnel. Higher enegy states (unfold proteins) search for different protein conformations with lower energy (protein intermediates) until they reach the natuve structure. It can follow different pathways. it depends on the environment (pH, solvent, interactions) but always is thermodynamically favorable. It can also lead to misfolding and aggregation.

Question 32

Hsp70 chaperone system

Answer 32

Hsp70 proteins (DnaK in bacteria) bind to hydrophobic regions in unfolded proteins during translation, while the polypeptides are coming out of the ribosome. They keep the protein in open conformation to aid its folding

Question 33

The multi-subunit chaperonin GroEL/GroES

Answer 33

GroEL/GroES form an isolation chamber for protein folding. - A single unfolded protein enters the cavity and emerges folded -> requires ATP. - protein folds in isolation. Hydrophobic regions are protected inside the chamber, avoiding aggregation. - The unfolded protein is captured by the hydrophobic regions of the entrance. GroES acts as a lid. Once folded GroES is removed and the folded protein emerges.

Question 34

Ubiquitination

Answer 34

In eukaryotes proteins are labelled by ubiquitin. Ubiquitination involves 3 steps: 1. Activation; the ubiquitin activating enzyme E1 uses ATP to bind ubiquitin to a cysteine. 2. Conjugation; transferred to the ubiquitin conjugating enzym E2. 3. Ligation; transfereed to the lysine of the target protein by an Ubiquitin ligase E3. Can be reversed by De-ubiquitinases (DUB)

Question 35

Proteasome

Answer 35

A protein complex that degrades ubiquitinated proteins in 2 short peptides. These can be used for: - degraded to single aminoacids to build new proteins - Antigen presentation for the immune system - Biologically active peptides.