Microbio Exam 3

What are the two important aspects of the genome of prokaryotes

• Base pairing machinery → gene expression and gene regulation

• Polarity

  • 5’ → 3’

  • position of carbon on sugar backbone

Genomes of prokaryotes are _____ and are comprised of _____ and _____.

haploid (one copy of genetic material)
chromosomes

megaplasmids

Where is DNA organized in the cell?

Nucleoid

How is DNA packaged in the nucleoid?

In a series of domain loops using anchoring proteins and supercoiling

Do prokaryotic bacterial cells have histones?

No, but has proteins similar to histones. This DNA is not packed as tightly and allows genes to be expressed easier

What are plasmids?

Small circular pieces of DNA that replicate freely and carry additional genes such as antibiotic resistance

Where are the three places we can find genes in a cell?

• Megaplasmids

• Chromosomes

• Plasmids

How can the sequence of an entire genomes be deduced? How is sequencing a bacterial genome easier?

Through fragmenting the DNA, sequencing and overlapping the sequences on a computer. Bacterial genomes are easier to sequence because their genome is so small.

What are contigs?

Continuous fragments of DNA that are lined up to overlap for a complete sequence

How are predictions made during genome annotation by DNA encoding genes?

Looking for Open Reading Frames (ORFs)

What is important for an ORF to have?

• If there is a ribosome binding site upstream

• Typically the longest and biggest

• If the ORF encodes a protein that is found in another organism

How do prokaryotic genomes differ from eukaryotic genomes?

Prokaryotic genomes are dense with little space in between genes. Much of the sequence is genes and very little segments of noncoding DNA

How can the functions of genes sometimes be predicted? What is an examples of this?

Because genes that are evolutionary related to each other will be similar to each other either within the same species or in different species. An example of this is speciation, all genes are related, they come from the same ancestral genes

What are motifs and why are they important? What is an example?

A conserved domain in one part of the protein that can give clues to function.

Ex. membrane-spanning domain, DNA- binding domain

What are some classifications of functional groups that genes in the genome can be classified into?

Cellular processes, energy metabolism, transcription

Explain how we analyze the genome of the organism that causes syphilis

It is missing many biosynthetic pathways, but has lots of transport systems, uses carbohydrates as its energy source, no TCA cycle or oxidative phosphorylation

What is gene expression? List the steps

The flow of information from the DNA to the final gene product

• DNA to mRNA through transcription

• mRNA to polypeptides through translation

What is read by RNA polymerase in order to produce mRNA?

The template strand of DNA

What are the two final products that gene expression can produce?

Proteins and RNA, not all genes encode proteins

What is mRNA and what is its function?

Messenger RNA that encodes protein

What is rRNA and what is its function?

ribosomal RNA that synthesizes protein as part of ribosome

What is tRNA and what is its function?

transfer RNA that shuttles amino acids to the ribosome

What is the function of catalytic RNA?

Carries out enzymatic reactions

How are bacterial genes organized?

As transcriptional units either contain a single or multiple genes in an operon

What is monocistronic RNA?

One gene is transcribed

Where does transcription start?

At the promoter

What is polycistronic RNA?

Multiple genes are transcribed

What does RNA polymerase do?

It is the enzyme that transcribes genes in bacteria, it has 5 subunits and a sigma factor

What is the purpose of RNA polymerase having a sigma factor?

The sigma factor allows RNA polymerase to bind where there are promoters

How do sigma factors work?

By recognizing DNA sequences called promoters, which indicate where transcription should begin, by binding to the -335 and -10 regions

What are housekeeping genes?

Genes that are always on and highly expressed

What are the +1, -10 and -35 regions on DNA sequences?

• +1 is the start site of transcription

• -10 is 10 base pairs away from the start of transcription

• -35 is 35 base pairs away from the start of transcription

Will the promoter sequence on each strand of DNA always match the average consensus sequence?

No, alteration of the consensus sequence can increase or decrease transcription because sigma factor binding gets stronger or weaker

Explain the example we saw with altering the lac promoter to make it match the consensus sequence?

When mutated to look the same as the consensus sequence, transcription increased and when altered to look different from the consensus sequence, transcription decreased.

What are 2 examples of different sigma factors that promoters will recognize?

Heat shock-induced genes, genes for motility and chemotaxis

What is a holoenzyme?

RNA polymerase with a sigma factor

Why might some genes contain multiple promoters that are recognized by different sigma factors?

May need a gene at different times / placed under different conditions

• Ex. Heat-shock genes are housekeeping so they are always on at low levels

  • There will be another promoter for when the cell is in heat-shock that will heavily increase expression

What does the initiation stage of transcription begin?

When RNA polymerase holoenzyme binds to a promoter and opens the DNA

Once RNA polymerase binds to the promoter, how does it form the open complex?

With rNTPs (ribonucleotides)

Once transcription starts, what happens to the sigma factor?

It is no longer needed and falls off

What is the elongation stage of transcription?

When RNA polymerase extends the growing RNA

During elongation, how does the RNA polymerase read and make RNA?

• Reading 3’ → 5’

• Making 5’ → 3’

What are the two types of transcription termination?

Rho-dependent termination and Rho-independent termination

Explain what Rho is and how rho-dependent termination occurs

Rho is a protein that binds to the RNA, when it comes in contact with the RNA polymerase, they will both fall off and transcription will end

Explain how Rho-independent termination occurs

Single stranded mRNA folds back on itself and base pairs to itself causing a hairpin loop. When RNA polymerase reaches this part of the sequence it causes termination.

What does the hairpin loop consist of?

GC rich area followed by many Us

What is rifampicin and how does it affect bacterial cells vs human cells.

A medically relevant antibiotic that targets bacterial RNA polymerase by blocking the exit tunnel. If humans are taking this antibiotic, our transcription will keep going

Describe the structure of a tRNA

They have an anticodon on one end and the matching amino acid on the other

How do the ribosomes read the mRNA to bring in tRNA complements?

5’ → 3’

What are the two subunits: large and small of a ribosome?

• Large: 50S

• Small: 30S

• The two units work together in translation

What are some other sites of a ribosome?

tRNA P site, tRNA E site and tRNA A site

How is the tRNA A site and the tRNA P site bonded?

Through a peptide bond that the ribosome creates

What does the small subunit of the ribosome do during the initiation stage of translation?

Binds to the mRNA at the start codon (AUG) and to IF1 (initiation factor 1). Then fMet-tRNA binds to the start codon connected to the small subunit. IF2 attached with GTP after

How can start codons be identified by bacterial ribosomes in translation?

Through base pairing between the rRNA and the Shine-Delgarno sequence just 5-7 base pairs upstream of the start codon

What are three common start codons found in bacteria?

• AUG

• GUG

• UUG

How to remember the 3 sites of a ribosomes?

EPA → environmental protection agency

What happens to the GTP that is attached to the invitation factor on the ribosome after the 1st amino acid is added?

It gets hydrolyzed and used for energy IF2 also falls off. The large subunit is then able to bind

What does it mean for a tRNA to be charged?

It has an amino acid on it and a high energy bond attached

When does termination of translation occur?

When the ribosome reaches a stop codon and release factors disassemble the complex

What is an example of a medically relevant antibiotic that targets the bacterial ribosome and how does it do that?

Inhibits tRNA binding because of distortion of the A site

How can transcription and translation be coupled in bacteria?

Because there are no organelles so DNA is not in the nucleus, it is free floating. Transcription and translation occur in the same time and place

How does each gene function in polycistronic mRNA?

Each gene has its own ribosome-binding site, start codon and stop codon, each protein can be translated separately

Once proteins are formed, what must happen and how does this process occur?

They must be folded into the correct tertiary structure, they are usually helped by chaperon proteins

Explain how chaperon proteins work

They bind to proteins right as they come off the ribosome and folded into an active form

What happens if the protein is not folded properly?

The protein can be fed to Growl for refolding, but this requires energy.

What are two examples of chaperone proteins

DnaK and DnaJ

If the protein still doesn’t fold correctly or unfolds due to hear what will happen to it?

A protease wil degrade it.

What are the two says in which proteins that need to be in the periplasm / outside of the cell or in the plasma membrane can eb secreted out?

Either posttranvslationally or cotranslationally

Explain the post translational SEC pathway

• Ribosome produces a protein

• Protein binds to SecB which transports to SecA

• SecA is bound to SecYEG

• SecYEG / SecA is a motor protein that uses ATP to push protein outside membrane

Explain the co-translational SEC pathway

• As the ribosome is producing a protein, SRP binds to the protein and pauses translation

• SRP carries the entire ribosome and the protein to SecA / SecYEG which uses energy to push protein out or into membrane

What is the main energy course of polypeptide chain elongation in the ribosome?

GTP

Besides ATP what are 4 other energy sources used for transport?

• Proton motive force

• GTP

• ADP

• Phosphorylation of substrate

Explain how gram-negative bacteria can use different systems to secrete proteins out of the cell:

• large strcutres that span from the plasma membrane → periplasmic space → outer membrane

• some structures have needle like structures that point out past the outer membrane which allows it to inject proteins into another cell

• these structures move proteins, toxins, protease, DNA, and virulence factor

• all systems / structures use energy

What is the Type III System?

• Virulence factors into host cells

• Used in pathogenesis

• chaperone brined protein to structure where it is injected out

• Uses energy

Explain how E. coli uses type III secretion system:

• Export their own receptor into host cells

• The receptor allows it a space to bind

• Sits on a pedestal of an epithelial cell

• E. coli typically not harmful unless it is the pathogenic strain

What genes besides metabolic genes are regulated (list 2)? Why do cells only express genes that they need?

• Cell cycle regulation

• Flagellum regulation

• Cells express only the genes they need to reserve energy

Explain positive regulation:

An activator will bind to the DNA that causes transcription

• When the activator is not present, transcription is not occurring

Explain negative regulation

A repressor will bind to the operator and transcription will stop

• when the repressor is not present, transcription is occurring

Activators are also called ______

postive regulators

Repressors are also called ______

negative regulators

Uptake and catabolism of lactose by e.coli requires what?

lac permease (symporter) and B-galactosidase

Explain the uptake and catabolic system of lactose by E.coli

• Requires Lac permease which is a symporter to import lactose and H+ inside the cell

• B-galactosidase breaks down lactose into galactose and glucose

• B-galactosidase can also convert lactose into allolactose which can also be brown down into lactose and galactose

What pathways will glucose and galactose directly feed into?

• ED pathway

• EMP pathway

• PPP pathway

What is the gene for lactose catabolism?

The lac operon and a regulatory gene with its own promoter located upstream

Explain the genes for lactose catabolism

• lacI has its own promoter that produces mRNA and translates to a lack repressor that binds to the lac operon and turns off expression (NOT PART OF LAC OPERON)

• LacZ, LacY and LacA are all part of the operon and controlled by the same promoter

What does lacZ code for?

B-galactosidase

What does lacY code for?

lactose permease

What does it mean that the lac operon is an inducible operon?

Transcription is normally off but when an inducer is bound, transcription will occur

What is the lac operon controlled by?

A negative regulator or repressor

What is occurring when no lactose is present?

• No transcription

• Repressor is bound but comes off and on all the time → small bit of expression

What occurs when lactose is present?

• allolactose acts as the inducer and binds to the repressor

• the repressor molecule changes conformation and can no longer bind to DNA

• transcription occurs

The lacZ gene has a mutation that changes a codon that normally encodes an amino acid to a stop codon, will lacY still be translated?

• Yes, but not translated well

• There is always a little bit of expression occurring so it will still be translated but not as good as it would if there were no stop codon present

What would be the phenotype of a cell where lacZ is deleted?

Lactose won’t be able to get broken down because no B-galactosidease will be made

What would be the phenotype where lacI is deleted?

LacI encodes for the repressor, so there will be no repressor made / bound so three will be constant expression

How can we fix the issue of the lac promoter being a weak promoter?

Adding a positive regulator or activator → CRP bound to cyclic AMP

• will give high levels of expression

How do CRP and cyclic AMP work together?

CRP binds to cyclic AMP and then form one unit that binds to RNA polymerase

How do the levels of cyclic AMP differ with glucose levels.

• High levels of glucose = Low levels of cyclic AMP

• Low levels of glucose = high levels of cyclic AMP

What happens when cAMP-CRP binds to DNA?

• DNA is bent and cAMP-CRP physically interacts with RNA polymerase

• RNA polymerase is bound to -10 and -35 regions

• This give high levels of expression

What is unique about the lac regulator binding regions?

There are specific DNA sequences that are for RNA polymerase, cAMP-CRP and the lac repressor

If E. coli cells are grown in the presence of both glucose and lactose, what happens?

• The cell will use all the glucose first

• There will be a lag phase to turn on lac operon

• lactose will be used second, slower rate than which glucose is consumed

• stationary phase after all lactose is used