Microbial Genetics Exam Two - Final

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What is the primary structure a protein?

A chain of amino acids joined by peptide bonds

What is the directionality of a protein?

N term – C term

What are the two basic forms of secondary structure?

Alpha helices and Beta sheets

How is the tertiary structure of a protein maintained in terms of hydrophobicity and hydrophilicity?

Hydrophobic amino acids inside, hydrophilic amino acids outside

What happens to a protein during heat exposure?

Denatures – bonds are broken (hydrogen bonds first – exposes hydrophobic regions which leads to aggregation)

What is the final conformation of a protein determined by?

The primary structure of each polypeptide

What do chaperone proteins do?

Bind to the proteins through hydrophobic regions and then allow the burying of hydrophobic regions to the core, allowing hydrophilic regions to be on the surface

What is Hsp70 also referred to in E. coli?

DnaK

What is the function of Hsp70 in proteins that denature?

They bind to hydrophobic regions in denatured and nascent proteins to keep the hydrophobic regions from binding to each other prematurely

How does Hsp70s ATPase activity help it?

It is required for the binding and dissociation of itself from the proteins it has helped/just folded

Can Hsp70 function by itself?

No, has cochaperones DnaJ and GrpE which are required to help regulate ATPase activity and cycling of Hsp70

What are other substitutes of DnaK?

Trigger factors (bind to proteins and help their folding), ClpA, ClpB, ClpX

Clps – function as chaperons; barrel like proteins; can also cause degradation of proteins

What is the structure of chaperonins?

Two barrel, two chamber structure that is a much larger and more complex chaperone compared to Hsp70

How do chaperonins such as GroEL work?

Unfolded protein goes into the hydrophilic barrel, then the lid (in this case, GroES) notices this and goes onto the barrel to close it causing the inner core of the chamber to go from hydrophilic to hydrophobic (making the protein send the hydrophobic to the core and hydrophilic residues to the surface, causing the unfolded protein to become folded inside the chamber); once folding takes place, lid comes off, and folded protein leaves; next unfolded protein goes into the other chamber

When are chaperonins induced?

During heat shock and other stresses

What are group I chaperonins?

Bacteria, mitochondria and chloroplasts of eukaryotes

What are group II chaperonins?

Archaea and eukaryotes; different protein sequences and different number of subunits

What type of channel is the SecYEG channel?

A secretory channel that is a heterotrimeric protein made up of SecY, SecE, and SecG

Which protein forms the channel of secYEG?

Sec Y

Is the inner channel of secYEG hydrophobic or hydrophilic?

Hydrophilic

Is the plug hydrophobic or hydrophilic in nature?

Hydrophobic

What is the function of the plug in secYEG?

It is normally closed, keeps traffic to the channel; highly selective for passage unless it is something MEANT to be transported through it such as a protein. The plug interacts with the proteins signal sequence, which is what allows it to open and allow the protein through

What is the function of a proteins signal sequence?

It is only a pass to get through secYEG; not needed for the proteins function and thus is removed during processing

Where is the signal sequence present on a protein and what are the different regions?

Signal sequence is present on the amino terminus; first 1-5 baes are basic n-region; h- region is hydrophobic region and that is 7-15 amino acids; c-region is next and that is the polar region; after those there is a cleavage site where a protease or peptidase can break the protein bond to get rid of the signal sequence leaving just the mature region

What is the function of secB?

It can bind to the target protein following translation completion; does not allow for it to bind yet; it keeps the protein unfolded until it binds to SecA. It brings the unfolded nascent protein to the sec channel and binds to the SecA protein, passing it off

What is the function of secA?

Hydrolyzes the ATP and pushes the protein (hydrophobic end first since that has the signal sequence) so that the plug will move away and allow the protein/polypeptide to go through the channel and get processed in the periplasmic space. In the periplasmic space, peptidases cleave the signal sequence allowing the mature protein to be folded into its tertiary structure

Compare the length of the protein in the cytoplasmic fraction of PCR and the periplasmic fraction of PCR after post-translation translocation.

The periplasmic/secreted fraction will be somewhat shorter

What is the process of co-translational translocation?

The Signal Recognition Particle (composed of FFH protein) which is a riboprotein complex, binds to the hydrophobic region of protein, bringing whole ribosome to dock onto the SecYEG channel with the help of protein FtsY that is also referred to as docking protein. During this, protein synthesis was paused, waiting to be docked. Once FtsY dissociated, the ribosome will resume protein synthesis. The first region that will hit plug, is hydrophobic region, allowing the plug to move. The protein will not pass through. YidC (insertase function) helps it to pass through a lateral channel, inserting it into the plasma membrane

What are transmembrane domains composed of?

Hydrophobic amino acids – there are seven of those

Why can transmembrane proteins not be translated in the cytoplasm?

Has many hydrophobic domains, would interact with many other hydrophobic regions and form aggregates

What type of proteins does the SRP pathway target?

Proteins that are to remain in the inner membrane

Where does this type of co-translational translocation happen?

 On endoplasmic reticulum surfaces in eukaryotic cells

What are the differences between prokaryotic and eukaryotic translation?

Coupled transcription translation occurs only in prokaryotes; The life span of RNA in prokaryotes is much shorter than in eukaryotes; Different ribosomes are used (70S present in prokaryotes, 80S in eukaryotes); Eukaryotic translation initiation region is initiated via Cap dependent, has Kozak sequence, and have IRES; Prokaryotes are monocistronic, eukaryotes are polycistronic; Different initiation, elongation, and termination factors; Prokaryotic translation is faster; the initiator amino acid is not formylated in eukaryotes

What are mutants?

Direct offspring of wild type members

What are alleles?

Different forms of the same gene - copies or duplication on the same DNA

What are useful phenotypes in bacterial genetics?

Colony color, size, smooth vs wrinkled, disappearance

What are auxotropic mutants?

They occur when genes that are required for essential growth substance are mutated

What are catabolic mutants?

When they can’t make essential catabolites or derive energy from a particular substance

What are conditional lethal mutations?

Mutations that cause lethality under one condition (nonpermissive) but not another (permissive)

What is the only genetic way to identify essential genes in haploid organisms?

Conditional lethal mutations

What principle does heredity in higher order organisms follow?

Darwinian principle – mutations arise randomly in species and if those mutations are desirable they would be naturally selective, then faithfully inherited in subsequent generations to become dominant member of population

What is Lamarckian theory?

Mutations are not created randomly, they are directed by the condition the species is residing in (ex. Toxic substance pushes evolution/mutation)

What did the Luria Delbruck experiments show?

Same culture started at same time showed each plated colony was roughly the same in terms of mutation. When there were multiple different tubes, each plate had different amounts and random amounts of mutations.

What did the Luria Delbruck experiments show

Same culture started at same time showed each plated colony was roughly the same in terms of mutation. When there were multiple different tubes, each plate had different amounts and random amounts of mutations. Thus the finding was that mutations occur randomly and are then selected by the environment rather than arising in response to selective pressure

Are the number of mutations and number of mutants proportional?

No.

How does overuse of antibiotics contribute to the emergence of resistant bacterial strains?

Antibiotics are not causing the resistance, but the mutations that occur are not wiped out by antibiotics and thus continue to propagate and populate, gaining numbers and becoming the predominant type of bacteria

How can a base pair mutation occur?

One base is paired incorrectly initially (ex. G gets paired to T) and not caught. Then during the duplication process, it splits and goes through replication. The G is paired correctly with C leading to a wild type copy, while the T gets paired with A leading to the mutant strand/copy. This can happen because sometimes thyamine is in an enolic form, leading to bonds being able to form between guanine and thymine since the nitrogen on thymine can donate a proton to base pair with the oxygen present leading to three hydrogen bonds forming.

What does transition mean?

A purine replaced by another purine, or a pyrimidine is replaced by another pyrimidine (T-A goes to C-G or C-G goes to A-T)

What is a transversion?

A purine goes to pyrimidine or pyrimidine goes to purine. (T-a goes to A-T or C-G goes to A-T)

How does a base pair change through deamination occur?

At a higher temperature, cytosine can be deaminated to Uracil; Methylated C is deaminated to T

What mutants show higher rates of spontaneous mutation?

Ung

What type of deamination cannot be recognized by ung?

Methylated C being deaminated to T

What is the repair mechanism for deamination?

Cytosine becomes deaminated, then uracil N-glycosylase removes uracil, the uracil base is cleaved off, and the DNA strand is degraded and resynthesized with cytosine at that position.

How does oxidation of bases lead to base pair changes?

Oxidative stress can modify G to 8-OxoG which can base pair with A, causing GC to TA or AT to CG transversion mutations

What are the consequences of base pair changes?

Changes in coding region (silent, missense, nonsense mutations)

What is a silent mutation?

There is no change in the amino acid sequence of the polypeptide (change in third wobble base of the codon)

What is a missense mutation?

One amino acid is replaced by another, which may have little to no effect if both amino acids have similar properties

What are nonsense mutations?

Changes that result in stop codons, and these are AKA chain-terminating mutations

What are base pair changes collectively referred to and why?

Point mutations as they map to a particular point in the DNA

What is a frameshift mutation and when do they occur?

They cause a shift in the reading frame, and they occur when one or a few base pairs are added to or removed from DNA; they are usually not leaky and almost always inactivate the protein

Can frameshift mutations be reverted?

They can; some bacteria take advantage of high frequency and reversion rate to avoid the host immune system

What is the cause of frameshift mutations?

Generally happens during DNA replication when the DNA complex is using template DNA strand to lay a new strand, and the template strand slips a little which can cause addition or deletion of one or two bases. It is facilitated by the presence of a short-repeated sequence

Present an argument how frameshift mutation can be more severe compared to mutation caused by base deamination.

Deamination just changes one amino acid whereas frameshift mutation changes the whole downstream sequence altering the whole protein

What are deletion mutations usually caused by?

Ectopic recombination between directly repeated sequences located in different regions of DNA.

What are direct repeats?

When two sequences read identical or similar in 5’-3’ direction

What is ectopic recombination?

Recomination outside the correct places

How does a deletion mutation occur in the same DNA molecule?

Repeated sequences in DNA end up “looped out” of the DNA molecule and thus are lost forever

What are the properties of deletion mutations?

Not usually leaky (loss is permanent and cannot be fixed); it can affect two or more genes; mutations do not ever revert; the naming of the gene is starting with delta

What do tandem duplication mutations result from?

Ectopic recombination between directly repeated sequences in different DNA molecules

What do tandem duplication mutations result in?

Duplication of a region immediately followed by its duplicate

When will tandem duplication mutations affect the gene and when may they not?

If tandem duplication occurs within a single gene, it will usually inactivate the gene. If it is long enough to include two or more genes, then it may not inactivate any genes

Are tandem duplications stable?

No, they are very unstable and revert at a very high frequency

How do inversion mutations occur?

The DNA sequence is flipped over by ectopic recombination and the recombination occurs between inverted repeats

Can inversion mutations be reverted?

Yes

What are the results of inversion mutations?

No phenotypic changes are caused, and no genes are inactivated since inversion junctions often occur in spacer regions

How are inversions named?

IN in capital then the length of genomic information that’s been flipped in brackets.

What are insertion mutations caused by?

Smaller insertions are caused by replication slippage that results in frameshift mutation. Larger insertions are caused by insertion elements spontaneously moving or “hopping” from one place to the other

What are insertion elements?

They are small, transposable elements that exist in multiple copies in many bacterial genomes, and they carry genes for enzymes to promote their own movement. They are in multiple copies so they also serve as sites for ectopic recombination that can result in deletion, duplication, and inversion mutations. They may also contain transcription termination sites.

Are insertion mutations leaky and do they revert?

They are usually not leaky and they rarely revert

What mutations are caused by genetic recombination?

Tandem duplication, inversion, deletion

What is the difference between reversion and suppression?

A true reversion would restore the original sequence of a gene, while suppression can cause restoration of a function that was lost through a second mutation elsewhere in the DNA (suppression mutation)

What are intragenic suppressors?

When one amino acid change makes a protein inactive can be rescued by another amino acid somewhere else in the protein that restores its activity (suppression of frameshift by another frameshift close by in the same gene)

What are intergenic suppressors?

Suppressor mutations that occur in other gene locations; they may restore the function of the originally mutated gene or provide another gene to take its place. Alternatively, it may alter another gene product that must interact with the original gene in a complimentary way. They may also restore the viability of a mutant by inhibiting the accumulation of a toxic intermediate

What is an example of a suppressor mutation that restored the viability of a mutant?

GalE mutation that can be suppressed by GalK mutation (galactose metabolism mutation in E. Coli)

What are nonsense suppressor mutations and how do they work?

A type of intergenic suppressor that is usually a mutation in a tRNA gene; it changes the anticodon so that the tRNA can recognize a nonsense codon. The mutated tRNA will still be charged by cognate enzyme with the xognate amino acid, allowing the synthesis of active polypeptide by suppressing the original nonsense mutation

What type of specificity do nonsense suppressors demonstrate?

Allele specificity – if one suppressor recognizes one codon, it can only suppress that one codon

What are the types of nonsense suppressors?

Tandem duplication of tRNA gene (unstable); more than one tRNA coding gene (stable); wobble factor (gives stable suppression without requirement of two copies of tRNA gene)

How are nonsense suppressors named?

UAG (amber); UAA (Ochre); UGA (opal)

What stop codons can a tRNA suppressor with anticodon 3’- AUU – 5’ recognize?

UAA and UAG

What are the consequences of nonsense suppression?

The coding sequence might end with more than one type of stop codons in failsafe mechanism; can affect mutations in essential genes or be unaffected if it’s a missense mutation; the race between suppressor tRNA and the release factor will occur to determine if producers will continue

Do eukaryotes allow for premature stop codons?

No, nonsense mediated decay systems destroy the transcripts with premature codons to avoid toxicity

What are the two types of general machinery for repair that have been identified?

Methyl-directed MMR which fixes minor distortion; NER which fixes significant distortions

What are the specific mechanisms of DNA repair?

Base deamination; VSP repair; 8-oxoG; Base alkylation; photoreactivation

How do we know that cells have a repair system?

We know for a various of reasons such as the existence of species generation to generation which shows DNA stability. Additionally there have been studies regarding cells continuous vs intermittent exposure of a DNA damaging agent which have shown that during the intermittent exposure they have been able to survive longer (and a longer period before cell death occurring) – cells with a repair system exhibit a “shoulder” suggesting repair attempt to fix lower-level damage

What are the deaminating agents?

Heat (culture condition or environment); hydroxylamine; nitrous acid

What are the conversions caused by deaminated bases?

A to hypoxanthine (can pair with C; G to Xanthine (can pair with C)

How are altered bases repaired?

There are over a dozen specific DNA glycosylases that are specific for altered bases – some remove only the modified base, while others (AP lyase) remove the base and cut the DNA backbone on the 3’-end of the missing base; AP endonucleases work in cooperation with glycosylases (if need be) to cut the 5’ side of the missing base leaving just the 3’OH group

What does Vsr Endonuclease do?

It can recognize the TG mismatch and make a break next to the T allowing for the T to then be removed

How does the VSP repair pathway work?

So, DNA cytosine methylases can methylate C in specific regions in a sequence specific manner, allowing for Vsr endonuclease to recognize the TG mismatch and remove the T. Then the strand gets resynthesized by repair DNA Pol I. There is a very specialized mechanism for CCWGG sequence and it usually repairs TG mismatches, and the Vsr repair system also interacts with the mismatch repair system

Where is vsr gene located relative to dcm gene?

Downstream