Lecture 18- Bacterial Structure & Function

Question 1

Describe structure of Bacterial ribosomes

Answer 1

• Supramolecular structures for protein synthesis
• contain rRNA and r-proteins\
• 70s, which split to 50s & 30s subunits
• The 50s (large)subunit contains ≈34 proteins and is comprised of 2 rRNAS (23S &5S)
• the 30s (small subunit contains 21 proteins and is comprised of 1 rRNA (16S)

Question 2

Describe structure of Eukaryotic Ribosomes

Answer 2

• ​Larger than prokaryotic ribosomes 80S (sedimentation rate)
• 80S split to 60s & 40s
• 60S contains ≈50 proteins & is comprised of 3 rRNAS (28S, 5.8S, 5S)
• 40S contains 33 proteins and is compromised of 1 rRNA (18S)

Question 3

Describe structure of Archaeal ribosomes

Answer 3

• Ribosome is 70S & splits to 50s & ​30S
• ribosomes contain 3 rRNAS in total (16S,23s,and 5S) & 50-70 proteins
• Primary structures of actual rRNA & proteins are closer to Eukaryotes than prokaryotes

Question 4

How many Archaeal r-proteins are represented in Eukaryotes, and are there any exclusive r-protiens shared between Archaea and Bacteria but not Eukaryotes?

Answer 4

All Archaeal r-proteins are represented in Eukaryotes and there are no exclusive r-proteins between bacteria and Archaea

Question 5

How many total Archaeal r-protiens are present in ribosomes?

Answer 5

50-70 depending on species

Question 6

Which RNA’s must interact with each other in the ribosome for protein synthesis?

Answer 6

All RNA’s (m,r,t)

Question 7

Briefly describe the four stage process of protein synthesis

Answer 7

• Initiation- Initiation factors(proteins) are used to start protein synthesis - either IFs or EIFs(for Eukaryotes)
• Elongation of polypeptide chain using Elongation factors EFs or EEfs
• Termination, involving release factors (RFs or ERFs)
• Recycling subunits- using Recycling factors

Question 8

explain decoding process for protein synthesis

Answer 8

Involves both Small & Large subunits

in SSU (decoding process) the major functioning sites are:

• m-RNA tunnel to conduct mRNA
• *Decoding center**- begins with the selection of the aminoacytl (A site) tRNA that corresponds to complement of mRNA (anticodon)
• tRNA binding sites (AminoactyltRNA Peptidebond Exit)

*peptide bond between 2 neighboring tRNA Happens at P site

In LSU- Peptide bond formation- major functional sites are-

• tRNA binding sites A,P, & E
• peptidyl transferase center (speeds up peptide bond formation )
• peptide exit tunnel

*​start codon is AUG, stop is UAG, UGA, and UAA

Question 9

What is the function of peptidoglycan that is contained in most bacterial cells?

Answer 9

• provides strength
• porous for small molecules

Disruption or cell wall structure or inhibition of synthesis causes cell lysis

Question 10

Describe the Gram positive cell wall

Answer 10

• one phospholipid bilayer
• lower lipid content compared to gram + because of no lipopolysaccharide
• thick peptidoglycan layer

​peptidoglycan polymer made out of monomers (PG monomers)

repeated units are cross-linked during cell wall synthesis to provide strength

Question 11

Describe cell wall synthesis of gram + bacteria

Answer 11

PG monomers contain

• N-acetyl glucosamine (NAG
• N-acetyl muramic acid(NAM)- pentapeptide attached to it

1. ​​Autolysins break glycosidic bonds between PG monomers & the crosslinking at point of growth
2. PG monomers are synthesized in cytosol & bind to bactoprenol (BP)
3. BP transports PG monomers across membrane & inserts them into existing PG
4. glycosidic bonds link PG monomers to existing PG chain
5. Transpeptidases form peptide cross links between rows & layers of PG by…
6. First removing terminal D-alanine from pentapeptides to form tetrapeptides
7. this provides energy to link pentaglycine bridge (glycine is the link)
8. transpeptidases cross link pentaglycine to D-alinine (4) to L-lysine(3) of other monomers to provide strength to cell wall
9. ​*L-lysine is not necessarily aa#3 and cross linking is different in gram -

Question 12

Which amino acid is the most abundant in peptidoglycan cell wall of gram +?

Answer 12

Glycine- ​because of the pentaglycine ​bridge

Question 13

How is the acid fast bacterial wall different from Gram+?

Answer 13

• stains poorly with regular die, However with Carbon fuchsin (stronger die) it stains well (retains red color)
• has a thin peptidoglycan layer linked to a layer of arabinoglactan , linked to an atypical membrane contaning mycolic acids , overlaid with polypeptide & glycolipids with acyl lipids–> additional strength to cell wall
• contains porins, which transport small hydrophilic molecules through the outer membrane like gram - however they contain less and ​longer pores –> less permeable

Mycolic acids & other glycolipids also slow down entry of nutrients causing slow growth–> more resistant to antibacterial agents and lysosomal components

Question 14

How is gram -be cell wall different than gram +?

Answer 14

• 2 cytoplasmic membranes
• Higher lipid content (lipopolysaccharide)
• Thinner peptiidoglycan cell wall
• D-glutamic acid instead of D-glutamine
• meso-Diaminopimleic acid in place of L-lysine
• D-alanine of one monomer(tetrapeptide) connects directly to meso-Diaminopimleic acid Of another monomer

Question 15

Describe the process of gram staining

Answer 15

• Fix bacteria on solid support by gentle heating(kills)
• add primary stain (crystal violet- or gram stain)
• add Mordant Gram’s iodine stain (increases affinity for primary stain)
• decolorizer, alcohol, and or acetone (gram - becomes colorless)
• counterstain safarin

​if gram + remain purple , if gram - pink