Lecture 5: Bacterial Genome Replication And Regulation 1 Flashcards
What is a nucleoside?
-the base, the sugar, and no phosphate group are present
What is a nucleotide?
-the base, the sugar, phosphate group are present
What amino acids base pair together?
- A=T
- GC with triple bond
The combination of this leads to formation of a major and minor groove in the helix
What nucleotides are present in RNA, and what does the strand look like?
- A,G,C,U
- mostly single stranded, but can be double stranded
These RNA subtypes are produced after the transcription of DNA.
- tRNA
- mRNA
- rRNA
- snRNA
- miRNA
- siRNA
What is unique about bacterial DNA when compared to that of eukaryotic?
- mostly circular, with a bidirectional replication fork
- begins at the replicon, which is also copied.
What is DNAa?
-component of E.Coli replication that initiates replication, and binds the origin of replication (oriC)
What is DnaB?
Helicase. Involved with primosome assembly and DNA primase activity
DNA Gyrase?
Acts like topoisomerases to relieve supercoiling.
- separates daughter molecules in final stage of replication
SSB protiens have what function?
They bind to the free ends of DNA to prevent reformation of the double strand
DnaC has what function?
- helicase loader; helps direct the helicase to the DNA template
DNA Primase has what function?
Synthesizes the RNA primer; is a component of primosomes
DNA Polymerase III holoenzyme does what?
Catalyzes the DNA synthesis during replication. Contains 3’—>5’ proofreading mechanism
DNA Polymerase I does what?
- removes RNA primers, and fills in the spaces that are left by removing the primer
Ribonuclease H does what?
- helps to remove RNA primers
Tus has what function?
-signals termination of replication
What does topoisomerase IV perform?
-separation of chromosomes upon completion of DNA replication
What materials does DNA Polymerase require in order to properly perform replication?
- Template strand in order to make a complementary strand
- DNA or RNA primer
- dNTPs
What 3 proteins form the core enzyme of DNA polymerase III
- alpha
- epsilon
- theta
The beta clamp has what function and is formed how?
- holds polymerase III to the template strand DNA
- formed by the gamma subunit and ATP hydrolysis. After the ring is formed by the two subunits, they generate an affinity for the alpha and epsilon subunits
The gamma complex of DNA polymerase III does what?
-catalyze ATP to chaperone two beta subunits to bind to DNA,
This is also referred to as the clamp loader
What is unique about the alpha complex of DNA pol III?
-has DNA polymerase activity
What is unique about the epsilon subunit of DNA pol III?
- this subunit acts as a 3’-5’ exonuclease; therefore has proofreading activity
What does the subunit tao do in DNA pol III?
- this subunit is flexible and helps maintain the two core enzyme and sliding clamps within a certain distance to the clamp loader, which will sit behind the helicase at the replication fork
What happens with DnaA at the replication fork?
- Dna A binds to oriC which bends and separates the two strands
After DnaA binds at oriC, what proteins bind to help separate the two strands of DNA even more?
- DnaB and helicase bind followed by SSB proteins to prevent reformation
What is the function of DNA Pol. I?
-removes the RNA primers and fills in the gap with DNA
Replication process ends when reaching the ter site. Catenated chromosomes are what, and how are they fixed?
- two chromosomes that have not separated.
- a double stranded break is introduced in one circle, the other passed out, and the nick is ligated. Successful separation of teh two strands
How do dimerized chromosomes form and how are they fixed?
- they form at ter sites where crossing over has occured, and prevents separation.
- XerCD recombinase is required to fix the dimerized chromosomes
Eukaryotes solve the ‘end replication’ problem by using telomerase to prevent chromosome shortening. What do bacteria do?
- the ends of the chromosome are disguised as hairpins, which appears as a pseudocircular strand of DNA.
- LEading strand synthesis occurs, and produces a dimerized chromosome.
- dimerized chromosomes are repaired with the use of XerCD recombinase
What is the best way to describe a gene?
- nucleic acid sequence that codes for a polypeptide, tRNA or rRNA
What is a promoter and what does it do?
- the recognition/binding site of RNA polymerase
- functions to orient the polymerase
Is the leader sequence every translated?
-no, it will be transcribed into mRNA, but will not be translated into amino acids.
What sequence on mRNA is important for initiating the translation?
Shine Dalgarno sequence
What amino acid initiates protein synthesis in bacteria?
> N-formylmethionine
What is the difference between polycistronic and monocistronic mRNA?
- poly: is an mRNA sequence that will encode for 2 or more proteins.
- mono: an mRNA sequence that will encode for only 1 protein
When is the sigma factor used in bacterial transcription?
- sigma factor directs the RNA polym. to the promoter
- 1st step of transcription initiation.
When does transcription leave the initiation stage and enter into the elongation stage?
- after 12 nucleotides have been linked together
- when sigma factor dissociates.
What function can rho do during transcription?
- can help the terminator sequence elicit termination at the end of a gene
What are the two types of termination processes that can occur for transcription?
- Rho-dependent
2. Factor independent
Describe factor independent termination of transcription.
- the RNA polymerase will encounter an ‘A’ rich region at which it will begin to pause. The pause allows enough time for mRNA to form a loop, straining the DNA:RNA hybrid and falls away.
Describe rho factor dependent termination.
- The rho protein binds to the rot site on mRNA moving towards the RNA polym.
- RNA reaches rho dependent region on DNA, causing a pause.
- Rho catches and inserts itself into the new rho region in the bubble and separates the DNA:RNA hybrid like helicase would.
What is a codon?
- Genetic code word 3 bp long
- Specifies and amino acid
- The anticodon on tRNA is complementary
What are nonsense codons?
These consist of three codons, that are mostly used at translation termination sites. They do not code for an amino acid, and cause termination of a sequence.
What is the wobble effect and what are the benefits of it?
- Provides loose bp that is less specific for the 3rd position of codons.
- The benefit is eliminating the need for unique tRNA for each codon
Whic part of the tRNA is responsible for binding the amino acid?
-3’ end
What is aminoacyl-tRNA synthetase?
- an enzyme that catalyzes the attachment of amino acids to tRNA.
- each one is specific for all tRNAs that may properly attach
What does the 30S subunit of RNA contain?
Contains the 16S rRNA ribosomal binding site.
- which binds to the shine dalgarno sequence.
- binds proteins needed to initiate translation and amino-acyl-tRNA
What specific unit is contained in the 50S RNA subunit?
- 5S rRNA and 23S rRNA
What is the 23S rRNA region responsible for?
- ribozyme that catalyzes peptide bond formation
What phases are required during elongation of a polypeptide chain during translation?
- Aminoacyl-tRNA binding
- Transpeptidation reaction
- Translocation
What are the different sites located in the ribosome for tRNA?
P site: binds the tRNA attached to the growing peptide chain
A site: binds the incoming aminoacyl-tRNA
E site: does not truly have a position, but briefly binds the tRNA before it leaves the ribosome.
What takes place in order to terminate protein synthesis in prokaryotes?
- A stop codon must be encountered
- Release factors aid in recognition of a stop codon
- The 3 RFs use GTP hydrolysis to release the ribosome
- eukaryotes only have 1 RF
What is the function of a molecular chaperone?
- Help fold nascent proteins
- Protect cells from thermal damage
- Transport proteins across membranes
What is a genome?
All DNA that is present in a cell or virus
What is the transpeptidation reaction important for?
- catalyzed by peptidyl transferase 23S rRNA
- interaction of amino acid in the “A” site and the polypeptide chain in the “P” site that adds the new amino acid
What events occur simultaneously during translocation?
- peptidyl-tRNA moves from A to P site
- ribosome moves down one codon
- empty tRNA leaves the P site
- -is GTP dependent
What portion remains in the polypeptide after splicing?
- exteins
What is the Sec-dependent pathway?
- major pathway for bacteria to transport proteins across the PM, and is energy requiring
What pathways do gram- negative bacteria use for material secretion?
- Sec pathway
- outer membrane passage requires Type I,II,III,IV,V systems
The Sec pathway consists of multiple proteins that all perform different functions. What are these proteins and their function?
- Y, E, G: form a channel in the PM for protein travels
- A: uses ATP to translocate the preprotein through the PM
- B: chaperone protein that maintains the translocation state of protein
What are type I secretion systems similar to?
-ABC transport systems
What are the primary secretions of type I secretion systems?
- endotoxins, proteases, and other proteins
Who primarily uses the Type I secretion systems?
- gram positive
- gram negative
- archaea
What is the Tat system?
-protein translocation system that moves FOLDED proteins across PM
What system works in conjunction with the Type II secretion system?
- Tat system
What is the type IV secretion system best at secreting?
- proteins
- secretes DNA from donor to recipient bacteria DURING conjugation
Where can Type IV secretion system be found?
- gram positive and negative systems
What secretion systems are unique to Gram negative bacteria that secrete virulence factors?
-Type II, III, V
What type of transport is type V?
-sec dependent autotransporter
Which gram negative secretion system form injectisomes?
Type III
In what ways can gene expression be regulated?
- transcription initiation
- transcription elongation
- translation
- -all can be affected in some way to alter what genes will be expressed or repressed.
