Micro Exam 3

what are three binding sites for trna in ribosome and what happens in each

Acceptor site; incoming tRNA which is bringing in next amino acid coded for by mRNA Peptidyl-tRNA site: tRNA holding on to peptide is in P site Exit site: tRNA is no longer charged; will exit; tRNAs are reusable

What is the role of chaperones in the cell? Describe the difference between the GroES/GroEL system and the DnaK/DnaJ system.

Chaperones are proteins that are required for many cellular proteins to fold properly, the production of these proteins is upregulated when the cell experiences multiple types of stress. Collectively these proteins are often referred to as Heat Shock Proteins (HSPs). GroEL and GroES DnaK and DnaJ

What does it mean when a cell is "competent"?

Competence: An organism that is capable of taking up DNA from the environment.

episome

Conjugation requires transferrable plasmids like the fertility factor (F- factor) plasmid. The donor cell is the F+ cell and the recipient is the F- cell. The F-gactor plasmid can integrate into the chromosome. An episome is a plasmid that can exist both as an i ntegrated chromosomal piece or an extrachromosomal plasmid.

What are some examples of antibiotics that affect translation?

Streptomycin: inhibits 70S ribosome formation Tetracycline: inhibits aminoacyl-tRNA binding to the A site Chloramphenicol: inhibits peptidyltransferase Puromycin: triggers peptidyl-transferase prematurely Erythromycin: causes abortive translocationFusidic acid: prevents translocation

What is the difference between the lagging strand in the leading strand? Why is there a lagging strand?

each replication fork has two strands: leading strand: replicatd continuously in the 5′-to-3′ direction. lagging strand: replicated discontinuously in Okazaki fragments DNA poly III cannot synthesize in the 3'→5' direction, the lagging strand is synthesized short segments

Understand how temperature can be used to regulate the transcription and synthesis of sigma H in E. coli .

Want to respond to heat shock; 42 degrees can cause heat shock (orange part); rpoH encodes sigma factor gene; transcribe this gene at all times; result of transcript is mRNA if translated becomes sigma factor; cell use temperature to allow regulation; not linear, complementary base pairs form in mRNA; regions happen where ribosome binding site is (AT 30 DEGREES); decreases translation of mRNA, rpoH made under 30 degree system gets degraded taken to die by chaperone proteinsAt 42 degrees: regions of complementarity held together by H bonds break and mRNA becomes linear; ribosome binding site no longer hidden; make a lot of rpoH; chaperones busy trying to make sure proteins folding properly and keep from accumulating in high temperatures; frees up rpoH gene end with functional RNA polymerase

Why would one need to repress the tryptophan operon?

When tryp repressor synthesized, naturally in inactive form, if lots of tryptophan in cell, tryptophan interact with proteins and bind to aporepressor, then bind to corepressor, bind to operator region, repress tryp operon

How do we artificially manipulate organisms to make them competent?

alter the cell using cacl2 to make cells chemically competent or use electroporation to shoot dna across the membrane

What enzyme attaches the AA to trna? Where is the AA attached to the trna? How does the enzyme recognize the correct trna

aminoacyl trna synthetase - need a unique one of these for every amino acid ???

dnaC (helicase loader)

helicase loader; some proteins wrap completely around one strand of DNA it needs something to hold it on there

ow is plasmid replication linked to chromosome replication?

Not tied to chromosome replication. Each plasmid contains its own origin sequence for DNA replication, but only a few of the genes needed for replication. Thus, even when the timing of plasmid replication is not linked to that of the chromosome, many of the proteins used for plasmid replication are actually host enzymes. Which host proteins are used depends on the plasmid

What is the core polymerase

proteins required to elongate an RNA chain

what is recombination

rearranging genetic material

20. What is a release factor and where does it bind?

recognizes the termination codon or stop codon in an mrna sequence

why would bacteria want to pick up foreign dna? hwo can this be dangerous

repair damaged genes; method od repair; competitive fitness

what does DnaA bi nd to? What happens after DnaA binds?

dnaA comes in and recognizes 9 base pair sequence in the oriC; it is a small protein that binds to the 9 mer sequence; this causes dna to bend where the proteins are binding; bending is important bc the bending is an AT rich region, the AT rich region separates facilitated by the structure of the DNA -Once the AT rich region denatures then the enzymes involved in dna rep can access the dna;

dnaA

dnaA comes in and recognizes 9 base pair sequence; a small protein that binds to the 9 mer sequence; this causes to bend where the proteins are binding; bending is important bc the bending is an AT rich region, the AT rich region separates facilitated by the structure of the DNA; now enzymes involved in dna rep cna access the dna

What is conjugation? What are the basic steps of conjugation ? (See figure 9.3)

donation of genetic information that requires cell-to-cell contact Requires one of the cells to have a transferable plasmid. In Escherichia coli there is a plasmid called the fertility factor (F factor). Conjugation: have to have cell to cell contact in transfer of plasmid between cells, one cell w plasmid and one without, then through -One of the cells has to have a transferrable plasmid -I have a donor cell that has a copy of the plasmid; it sends out a conjugation pilus (yellow); it randomly goes out in different directions and eventually attaches to something and pulls that other cell towards it; the conjugation pillus retracts and brings the two cells in -All the information to bring the relaxosome is on the plasmid -They build a relaxosome between the 2 cell and through rolling circle replication (do a nick, thread one of the two strands of the molecule into this cell, other stays in the first cell, this cell uses this to make a complementary copy and maintain an intact plasmid, the other cell gets a singles trand and makes a complementary copy); after this is done; both cells have a copy of that plasmid -Started out w just one cell had it and now both have an identical plasmid -That requires a transferrable plasmid that has all the ifnromation to build the relaxosome; that also has other genes like antibiotic resistance gene

. What is the role of DNA gyrase in replication?

what prevents the buildup of torsional stress is the DNA gyrase that is located ahead of the fork, removing the positive supercoils as they form.catalyzes the formation of negative supercoils that is thought to aid with the unwinding process

What is semiconservative replication?

when u t ake pre-existing dna molecule a nd open it up for replication; each one of these two strands act as a template for synthesis of new strand;

What is a genomic island

§Genomic islands provide evidence for horizontal gene transfer. §These include: -Pathogenicity islands -Symbiosis islands Fitness islands

What is a transposable element? How is it different than a plasmid? What is the difference between an insertion sequence and a transposon? What is a transposase? What are the two types of transposition?

§Transposable elements move from one DNA molecule to another -Exist in virtually all life-forms -Can move within and between chromosomes §Insertion sequence (IS) -Simple transposable elements containing a transposase gene, flanked by short inverted repeat sequences -Are targets for the transposase enzyme -Transposons are complex transposable elements carrying additional genes -Moble genetic elements; have quite a few in human genome; a piece of dna that gets out and runs over and inserts itself back in; cannot move between cells but between chromosomes or in a chromosome -Multiple times of mobile genetic elements:Inserttion sequences; most simple; has inverted repeats; those inverted repeats flank a transposase gene;

What is the role of methylation in DNA replication? How does the concentration of DnaA affect initiation of replication?

*peep seqA slide* Immediately after a cell has divided, the level of active DnaA (DnaA bound to ATP) is low, and the inhibitor protein SeqA binds to oriC

. How are plasmids transmitted? Can all plasmids be transmitted from one cell to another?

- -The process of conjugation; two cells come in close contact w each other build something called a relaxosome and during rolling circle replication; the one strand is fed thru is fed through a relaxosome so it goes into the cell that didn't have a plasmid; now that cell has the information it needs to synthesize the second strand; From aleah: Some plasmids are self-transferable via conjugation, a process that requires cell-to-cell contact to move the plasmid from a donor cell to a recipientOther plasmids are incapable of conjugation (nontransmissible)A third group can be transferred only if a self-transferable plasmid resides in the same cell. In this case, the conjugation mechanism produced by one plasmid will act on the other plasmidAny plasmid released from dead cells can also be taken up intact by some bacteria in a process called transformationFinally, plasmids can be transmitted in nature by accidentally being packaged into bacteriophage head coats—in other words, by bacteriophage transduction

okazaki fragment

-: cant start my job until helicase is unwound about a 1000 nucleotides; it inserts primer and then primase inserts a primer and then I can use that template strand and synthesize my complementary strand; as im synthesizing in one direction the dna still continues to unwind in the other direction; after I finish synthesizing this segment I disengage and move to another primer; and start synthesizing until the 5' to 3'; keeps going this until it encounters its first primer; these are called okazaki fragments -They leave the sliding clamp so that the cell knows to go back and replace those rna primers; now dna poly 3 has finished synthesizing okazaki fragments;

What is catabolite repression?

-If the cell is concerned about the presence of glucose if glucose and lactose are both present we should get basal level of transcription -Cell has evolved to not let lactose into t he cell bc If i have glucose i don't want lactose -How do I keep lactose from coming in the cell if its outside t he cel? The way the cell can tell if theres glucose is the state of the PTS system; as soon as PTS gets a phosphate I slap it on glucose and glucose goes in the cell; essentially if im an active PTS system I dont hold on to phosophate very long; if the PTS system is not phosphorylated that tells me glucose is in the cell; if glucose isn't in the cell those proteins will be phosphorylated; I can use the PTS system to block the Lac Y permease; when IIA is nott phosphorylated there is glucose in t he cell so it blocks Lac Y permease; as long as those aren't phosphorylated 2A blocks permease; when phosphate starts accumulating I now t heres no glucose in t he cell so II A doesnt block Lac Y ??? Something about allolactose -Confused a bou the g raph

Do all proteins have the same half - life? What part of the protein plays a role in determining the half - life of that protein?

-If u look at sequence of polypeptide u can see what half life of protein is; the n terminal amino acid provides an internal clock that determines how long a protein will be around

Dna poly 3

-Polyermases build polymers; -Dna poly 3: If I have a single strand of dna; this is the template; if I just give dna poly the template it cant do that - it has to have a primer -Has to have a template strand, a primer, and only synthesizes in the 5' to 3' direction

hat is a protease, and why is this important for the cell?

-Proteases job is to cleave amino acid and degrade proteins

What are the two types of recombination and what are the differences between the two?

-Recombination is essentially m oving foreign dna to another dna molecule of some kind; basically dna that was not there before gets inserted into a molecule that's also made of dna -The two things have to be similar; theres usually some sequence similarity that stimulates t his process Two different DNA molecules in a cell can recombine by one of two main mechanisms: 1. Generalized recombination requires that the two recombining molecules have a considerable stretch of homologous DNA sequences. 2. Site-specific recombination requires very little sequence homology between the recombining DNA molecules. But it does require a short sequence recognized by the recombination enzyme; §Does not utilize RecA, moves a small amount of information. §Short segments of homology §These are recognized specifically by dedicated enzyme systems, which catalyze a crossover between them to produce a cointegrate molecule §Examples: The integration of phage lambda

structure of a ribosome

-Ribosome Is made up of amino acids and proteins •The structure of a ribosome -Large subunit -Small subunit •The activity within the ribosome -Acceptor site -Peptidyl-tRNA site -Exit site Translation occurs within the ribosome which is constructed from proteins and ribosomal RNA (rRNA) The structure of a ribosome Large subunit: 50s Small subunit (30s in bacteria)Come together and form one 70s functional ribosome

Rolling Circle Rep

-The dna from a bacterium has been gently released from a cell; small circular dna molecules calls plasdmids also exist; repA binds to the oriC; repA Is encoded by a plasmid (the gene that encodes for repA is stored on the plasmid); it holds on to the 5' end of the strand, repA knicks one of the strands and then it removes one of the 2 strands leaving a single strand behind); as replication continues the nick strand peels off until replication of intact strands are complete; the two ends of the nicked single stranded is joined together by repA and released; dna ligase seals the nick in the double stranded molecule; the single stranded dna can now be replication; a region becomes looped? Allowing rna poly access to form a primer bc I don't have any existing 3' OH groups;

What is the function of tmRNA?

-Tmrna acts as a trna and an mrna; only employed in certain situations; -If this is floating around the cell; heres my start codon and heres my stop codon; I need this part of the message to stay intact hwoever sometimes it might be cleaved; so now the ribosome doesn't know its broken -The ribosome recognizes shine Dalgarno sequence; so it starts translating but it gets up to the point where it broke and then it just sits there bc it has no info past where its broken bc theres no release factors bc theres no stop codons in thte A site -If that happens it sends out signals saying its stuck so this molecule docks here and artificially carries a message with it; essentially the message carries with it is called a degradation tag; -This allows the maino acid to release its peptide; but the problem is its not finished; its probably gonna misfold bc it doesn't have all the actual information -Instead of finishing this it tags it w a degradation tag -But by using the tmrna u can recycle ribosome and trna u just destroyed the message bc lots of info is missing -Once the polypeptides are made were not necessarily finished w the process, by nature of the genetic code every single polypeptide synthesized starts with methionine; -After synthesis is complete u have enzymes that come around and can remove the formular methionine eand make it regular methionine or just remove it altogether -

sliding clamp

-a protein that keeps everything together; 2 strands act as a template; the dna poly 3 replicates one of the strands and the other dna poly 3 uses the other as a template; they are tethered together by the clamp loader so that one doesn't get too far in fron t of the other -they don't actually synthesize dna but they stabilize the polymerase on the double stranded dna; if im dna poly I can bind here and I can add my c and t and a; ocassionally I fall off; and something has to load me back on there - turns out this is a rly inefficient way - so this is a secondary protein which stabilizes the dna poly; stabilizes the interaction between dna poly and its template

single strand binding protein

-bind single stranded dna; dna is normally in the cell as a double strand; the cell knows this is a bad thing; nucleases encounter single stranded dna and attack it; the cell doesn't want these to be attacked and don't want the complementary strands to come back together so the ssb keep them separated and protected

seqA

-binds to hemimethylated DNA; replication requires dnaA tot bind to the origin of rep; what if dnaA cant bind? We cant get replication; something else is binding there instead; binds to hemimethylated - one strand is methylated and the other is not; if I had just gone thru a round of replication u have old strand here and new strand here; in this case enzyme adds methyl groups and this helps it to tag its dna as itself; if I bing to a round of replication the old strand is methylated; but the enzyme has not been able to label the other half?? seqA has a rly high affinity for this; it binds to these sequences (same sequences dnaA wants to bind to); a methylase comes in and adds methyl group - then seqA falls off and dnaA can bind -Cell doesn't want to start new replication till it knows how the other round went -Once the AT rich region denatures then the enzymes involved in dna rep can access the dna;

Levels of expression of Lac Operon

1.Repressed 2.Basal-level 3.Activated -basal-level: if I don't have an activator or repressor active; I have no regulatory proetins; the strength of the promoter controls level of expression; f or lac operon the promoter Is weak - t he promoter keeps expression levels low; - -Lactose Present + Glucose Present; repressor is inactive; activator is inactive (glucose is present so Im not making cAMP; that's the inducer; my activator is inactive); basal level -Lactose absent + glucose present: repressor is active; activator is inactive; repressed -Lactose absent + glucose present; repressor is active; activator is active; repressed -Lactose present + glucose absent: repressor in active; activator is activel activated

How many DNA polymerases does E. coli have? Are they all involved in DNA replication?

5; DNA polymerase I (pol I): Acts on lagging strand to remove primers and replace them with DNA. Has 5ʹ-to-3ʹ exonuclease activityDNA polymerase II (pol II): Involved in DNA repair processesDNA polymerase III (pol III): Main replication enzyme; Commonality: All 3 have 3ʹ-to-5ʹ exonuclease activity - proofreadingonly DNA polymerase I and III primarily

Elongation - rep

??

Termination - rep

??

Whole process of dna rep

???

What is a restriction endonuclease? How do these he lp protect cells? How does a cell keep it's own DNA protected from restriction endonucleases?

???? Restriction endonucleases cleave DNA at specific recognition sites, which are usually 4 to 6 bp and palindromes. May generate blunt or staggered ends -restriction Enzymes produce enzymes in order to cut dna in certain location -Restriction endocnucleases cut at sequences that are usually palindromes -Different organisms have different restriction enzymes: the most famous one is isolated from ecoli; EcoRI; first RE that was isolated

Understand what a mutation is and that a mutati on may be harmful, neutral or beneficial? Know what the different types of mutations are. Point mutation Insertion/Deletion Inversion Reversion

A mutation is a heritable change in the DNA. Mutations can come in several different forms: oPoint mutation: change in a single base oTransition: purine → purine or pyrimidine → pyrimidine oTransversion: purine ↔ pyrimidine oInsertion (addition) and deletion (subtraction) of one or more bases oInversion: DNA is flipped in orientation Reversion: DNA mutates back to original sequence -Transversion are more severe than transition -Sometimes dna poly 3 accidentally incorporates the wrong base - point mutation -If it gets caught up on a difficult stretch to copy -inversion: part of the dna gets cut and then it flips over and reinserts itself -Reversion: if I had an A à t --> A that's a reversion

Repressors

Actions of a repressor: Regulatory site may overlap the promoter site Block the closed to open complex; Interfering with the polymerase or stabilizing the DNA; Effect: Transcription is repressedTwo ways to control repressor1. Repressor binds and works as roadblock; overlaps with promoter; this blocks promoter; if holoenzyme cannot see promoter, won't transcribe; results in little to no transcription; occasionally it will fall off and a few transcripts will occur but it'll bind again Inducer: To get repressor out of way, ligand binds to protein and changes protein conformation; now DNA binding sites are no longer available; repressor falls off; inactive because ligand bound to it (inducer, induces transcription = ligand); used when talking about catabolic pathways2. Have repressor protein, in inactive form floating around; to activate it, ligand (corepressor) binds to it and has opposite effect, now DNA binding sites are available and it can bind to regulatory protein and will repress; used in biosynthetic pathway

Activators

Actions of activators: Regulatory protein that increases levels of transcription; can transcribe without activator, just makes it faster; an inducer (ligand) binds to inactive activators, turns them on; doesn't overlap with promoter, further upstream; make RNA polymerases job easier; may increase affinity, facilitate denaturing of DNA to two single strands, etc.; means higher levels of transcription Enhance the activity of the RNA polymerase How do they do this? Accelerate transition to the open complex Alter the torsion of DNA facilitating melting; Cooperativity by increases the affinity of the polymerase for the promoter

How are bacteria able to regulate gene expression?

Activators Repressors

What is a plasmid? Which organisms have plasmids?

Archaea, bacteria, and eukaryotic microbes Two kinds of extragenomic DNA molecules can interact with bacterial genomes: Plasmid-encoded functions can contribute to the physiology of the cell. For example, antibiotic resistance -Circular molecule smaller than genome; have a lot fewer genes; don't have genes necessary to live -Genes on plasmid can provide physiological benefits -Ex: gene for antibiotic resistance; if u have a plasmid and it has an ampicillin resistant gene that could help u if u were xposed it; on plasmids gene can be beneficial under some conditions but not all the time -plasmids Plasmids are much smaller than chromosomes. Found in archaea, bacteria, and eukaryotic microbes Usually circular Need host proteins to replicate

How are Archaea; genomes similar to bacteria? similar to eukaryotes? unique?

Archaeal genomes combine features of bacteria and eukaryotes. Like bacteria, archaea have:-Polygenic operons-Asexual reproduction-Cells lacking a nuclear membrane-A single circular chromosomeIn most species of archaea, however, the processes of DNA replication, transcription, and translation more closely resemble those of eukaryotes Size: In general, eukaryotic genomes are larger than those of bacteria. End Replication Problem: Because their chromosomes are linear, eukaryotes require a reverse transcriptase called telomerase to replicate their ends. Eukaryotic cells pack their DNA within the nucleus using proteins called histones. A large portion of eukaryotic chromosomes are composed of noncoding DNA: Introns and pseudogenes

In what direction is DNA synthesized?

Birdirectional - u can watch it thru fluorescent microscopy; have a single genome w an origin of replication and termination; in prokaryotes there is one location for origin; in eukaryotes there are multiple origins; u start at the origin of replication and one replication fork moves clockwise around the genome and the other one moves counter clockwise around the genome --> we can finish replication in half the time 5' to 3'

dnaB (helicase)

DnaB (helicase) will be loaded onto the exposed replication forms unzips the dna; goes thru the entire molecule and moves thru there and the segment of dna in front of it breaks hydrogen bonds; first thing is to further denature the molecule; need a helicase for both replication forks

What is the purpose of having multiple sigma factors?

Each sigma factor helps core RNA polymerase find the start of a different subset of geneshelps the core enzyme detect the promoter, which signals the beginning of the geneSigma (σ) factors are the proteins that guide RNA polymerase to the beginnings of genes gene. A sigma factor first binds to RNA polymerase through the beta and beta-prime subunits. The bound sigma factor helps the core enzyme detect a specific DNA sequence, called the promoter, marking the beginning of a genepurpose: A single bacterial species can make several different sigma factors. Each sigma factor helps core RNA polymerase find the start of a different subset of genes

What is inducer exclusion and how is it related to glucose vs. lactose metabolism?

Failure of lactose to induce lacZYA during growth on glucose is due mainly to the fact that growth on glucose keeps lactose out of the cell. This phenomenon is known as inducer exclusion. If lactose cannot enter the cell, the lacZYA operon cannot be induced. The key to inducer exclusion is that a component of the glucose transport system, while transporting glucose, will bind to and inhibit LacY permease. The PTS transfers a phosphate from phosphoenolpyruvate (PEP) along a series of proteins to glucose during transport. When glucose is present, the glucose transport proteins continually transfer phosphate to glucose and, so, are usually left without phosphate. Unphosphorylated enzyme IIA interacts with LacY in the membrane and inhibits LacY activity. So, lactose cannot enter the cell, and the lac operon remains uninduced. When glucose is not present, the PTS proteins remain phosphorylated; enzyme IIA-P does not interact with LacY, so LacY transports lactose into the cell; and the lac operon is induced

hat is RecA?

Generalized recombination requires a protein called RecA RecA molecules are also called synaptases They are able to scanDNA molecules for homology and align the homologous regions

What is a transformasome? What type of bacterium makes a transformasome? Do they t ake in double or single - stranded DNA? What is the role of competence factors?

Gram-positive bacteria transform DNA using a transformasome complex. -If a cell can take up foreign information its called competency -They build something called a transformasome that's a protein that allows it to bring in foreign dna from the environment -Only make one if its beneficial for them to do so -Induced competency: its making cf's and excreting them outside the cell; these stand for competence factors; chemical signals; the reason they are sent out is basically bc it's a communication signal -If other organisms are doing the same thing then the number of cf's in that environment increases; -A cell can detect that cf;s are being secreted; if its just detecting its own the concentration doesn't get high enough for it to hav a response; but if more build up the cell detects the presence of other competence factors and the cell starts transcribing the genes necessary to createa transformasone -Doesn't do it unless other cells are around -Why would it wanna do this only if other cells are around? A transformasone picks up foreign dna; that increases the change that foreign dna is available for me to take in •Gram-negative bacteria transform DNA without the use of competence factors (CF). Either they are always competent, or they become competent when starved. •Transformation in most Gram-negative species is sequence specific. •limits gene exchange between genera

Three steps of rep

Initiation Elongation Termination

3 steps of translation? what happens in each step? how are trnaslation factor involved? what does if3 do

Initiation •Shine-Dalgarno Sequence -The upstream, untranslated leader RNA contains a purine-rich sequence with the consensus 5′-AGGAGGU-3′ -There is the s mall subunit of my ribosome, -IF3 binds to small subunit of the ribosomea nd keepst he large from associating ith t he small subunit until we are ready to start translation; facilitates t he bdindng of r na to subunit; -IF2 is gonna be associated with thet rna -IF1 binds to the s mall subunita t a unit called the A site where incoming trna's usually bind; taking out the parking spot so no trnas come in yet -The trna enters the p site by being escorted to if2, this is the only time it starts in the P site (only the first) all subsequent ones enter t he A site -The anticodon of the trna is complemenetary to the AUG start codon -Once t he initator trna is in place if3 is released, then the 50s subunit can dock, this triggers the hydrolysis of gtp on if2 -Now we elongate: 1. first eftu associated with gtp binds tot the free charged aminoacyl and brings it intot the a site, correct selection of the t rna complex is based on codon/anticodon paring, in thes econd step the ribosomes peptidoyltransferase activity catalyzes a peptide bond between the amino acid in the a site and t he one in the p site, now the 2 separate amino acids area ttached, went f rom indiivdial amino acids to a dipeptide, s imultaneously gtp is hdyrolyzes -Now ihave t o g et t he n ext codon into t he A site, ribosomen eeds to move forward 3 subunits -An elongated factor called EF-G is associated with GTP, ???? WATCH T HEVIDEOONT HIS

Three phases of transcription and what happens in each phase

Initiation: RNA pol holoenzyme binds to the promoter-This is followed by melting of the helix and synthesis of the first nucleotide of the RNAElongation: the RNA chain is extendedTermination: RNA pol detaches from the DNA, after the transcript is made

What is the stringent response? What role does ppGpp play in the stringent response? What synt hesizes ppGpp?

Lack of amino acids will trigger this response; means we cant synthesize proteinsUncharged tRNA enters A site, signals theres a problem; ReIA involved with ppGpp??Idling ribosomes stimulate the production of ppGpp; start interacting with RNA polymerase; decreases affinity of RNA polymerases for tRNA and rRNA genes, wont transcribe them because you can't use them right now because no amino acids to translate; ribosomal proteins still there because can regulate own transcription, have overabundance of ribosomal proteins because too few rRNA for them to bind to; build up in cell, cell wants to stop making proteins because building up and not being used and resources wasted; can bind to own polycistron, causes ribosomal protein to be repressedDecreases the affinity of RNA polymerase for promoters of rRNA genesBinding of ppGpp to RNA polymerase decreases synthesis of rRNA, which slows the rate of new ribosome synthesis. The overall rate of translation, then, will match growth rate.

What is the difference and the advantages of being a high-copy number plasmid vs. a low -copy number plasmid?

Low-copy-number plasmids segregate equally to daughter cells. High-copy-number plasmids segregate randomly to daughter cells. - Low copy number: when the plasmids in the cell u retain just one or two copiese or Hundreds of thousands (high copy) -If ur a low copy number theres a bigger risk that ur not gonna get inherited when the cell divides during binary fission -If ur a high copy number ur gonna segregate reandomy -Low copy number has to make sure plasmids gets separated to opposite poles during binary fission If the plasmid is not beneficiail its not worth the trouble so the yell can lose plasmids over time

How do plasmids replicate?

Plasmids can replicate in two different ways: 1. Bidirectional replication Starts at a single origin and occurs in two directions simultaneously 2. Rolling-circle replication Starts at a single origin and moves in only one direction

How does the two component signal transduction system operate? What are the components and what is the function of these components?

Popular in bacteriaMicroorganisms must be able to detect the conditions of the environment, and respond to changes in the environment.Systems meant to pass messages on; key to organisms sensing environment and having correct responseSense iron/phosphate availability, nitrate, etc. ENVIRONMENTAL SIGNALEach two-component system will regulate a different set of genes. The first protein in each relay, the sensor kinase, spans the membrane. A kinase transfers a phosphoryl group from ATP to a protein. The sensory domain of most sensor kinase proteins contacts the outside environment (or periplasm), while the other end (the kinase domain) protrudes into the cytoplasm.Each sensor protein of a two-component system recognizes a different molecule or condition. Once activated, the external sensory domain triggers a conformational change in the kinase domain that activates a self-phosphorylation reaction. Phosphate from ATP is attached to a specific histidine residue located in a different part of the protein. Then, like two relay runners passing a baton, the phosphorylated sensor kinase protein passes the phosphate to a cognate (matched) cytoplasmic protein called a response regulator. This transfer, called transphosphorylation, occurs at a specific aspartate residue within the response regulator. The phosphorylated response regulator commonly binds to regulatory DNA sequences in front of one or more specific genes and activates or represses expression

How does regulation of an anabolic pathway differ from that of a catabolic pathway?

Repressing biosynthetic pathways is fundamentally different from repressing catabolic (degradative) systems. Repressor proteins that control catabolic pathways, such as lactose degradation, typically bind the initial substrate or a closely related product (for example, allolactose in the case of the lac operon). Binding the substrate decreases repressor protein affinity for operator DNA. Thus, increased concentration of the substrate or inducer actually removes the repressor from the operator and derepresses expression of the operon. This derepression makes sense because the cell "wants" to make the enzymes that use the substrate as a carbon and energy source.In contrast, genes encoding biosynthetic enzymes are regulated by repressors (called inactive aporepressors) that must bind the end product of the pathway (for example, tryptophan for the trp operon) to become active repressors. The pathway product that binds the aporepressor is called a corepressor. Binding of the corepressor (end product) to the repressor increases the repressor's affinity for the operator sequence upstream of the target gene or operon. As the concentration of end product, such as an amino acid or nucleotide, increases in the cell beyond what is needed to support growth, the cell will shut the biosynthetic pathway down and not waste energy making a superfluous pathway or compound.

. What type of information is contained on a plasmid?

Resistance to antibiotics and toxic metals Pathogenesis Symbiosis Plasmid-encoded functions can contribute to the physiology of the cell.

what are the two types of transc termination? understand what happens in each

Rho-dependent: Relies on a protein called Rho and a strong pause site at the 3′ end of the geneRho-independent: Requires a GC-rich region of RNA, as well as 4-8 consecutive U residues

what are some examples of antibiotics that affect transc

Rifamycin B - binds to bacterial rna poly and inhibits transc initiation actinomycin d - non selectively bins to dna and inhibits transc elongation

Understand the categories of mutations. Silent mutation Missense mutation Nonsense mutation Frame shift

Silent mutation, nucleotide change that produces a codon that encodes for the same amino acid. Missense mutation, nucleotide change that produces a codon that encodes for a different amino acid than the original codon; -If u substitute hydrophobic for hydrophobic amino acid maybe ist not as big of a d eal -Usually bigger if its one from group of AA's to another -This is sickle cell anemia; ctt is cat so when u transcribe it you get gua instead of gaa, now gua encodes for valine (negative chart) instead of glu (hydrophobic), valine does not act well w water which affects the way the protein folds, Nonsense mutation, nucleotide change that produces a stop codon mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotidetrabso

how do mutations arise

The ability of DNA Polymerase III to proofread its work decreases the number of spontaneous mutations due to misincorporation. IF YOU CHANGE THE CHEMICAL PROPERTIES OF A BASE THEN YOU CHANGE ITS BASE PAIRING PROPERTIES!!! Mutations do occur 1. Tautomeric shifts in DNA bases that alter base-pairing properties 2. Oxidative deamination of bases 3.Formation of apurinic sites §Mutations can be caused by mutagens: §Chemical agents -Base analogs -Base modifiers -Intercalators §Electromagnetic radiation -X-rays and gamma rays: break the DNA -Ultraviolet rays: form pyrimidine dimers -Dna poly 3 has activity to catch mistakes; but sometimes it makes mistakes -Base analogs: look like a nucleotide so dna poly grabs then; but don't act like a nucleotide; -Modifiers: cigs have these, can attach to dna and form a glob -Intercalators: weasel ay into double strand similar to nucleic acid and can malform molecules -Uv rays - extdnsive damage to dnaa nd can result in things like skin cancer X rays and gamma rays - take doublestrand and essentiallycause doublestrandedbreaksand now have parts that are no l onger connected

Lac Operon from study guide

The lac operon is regulated by both a repressor and an activator. The lac operon contains genes that are necessary for lactose metabolism lacZ (β-galactosidase) , lacY (lactose permease) and lacA (Thiogalactoside transacetylase) . The lactose permease is a transmembrane protein that transports lactose into the cell. β-galactosidase has two functions the first being it cleaves lactose into glucose and galactose which can feed into the glycolytic pathway, and secondly it isomerizes lactose into allolactose (which is a ligand for the lac repressor). The role of thiogalactoside transacetylase is unknown. Lactose absent (Transcription is repressed) The repressor (LacI repressor) will be bound to two operator regions located near the operon. Binding of the repressor blocks the promoter and RNA polymerase will not transcribe the lacZYA genes. It is worth noting that occasionally the repressor will fall off allowing a very few transcripts to be made. Lactose present Allolactose, produced from lactose by β -galactosidase, binds to the LacI repressor removing it from the operator region. The genes ( lacZYA ) are now transcribed at higher levels because RNA polymerase is no longer blocked from the promoter. Low-energy levels (Transcription is activated) When a cell's energy state is low cAMP is produced in response. cAMP will bind to the cAMP receptor protein (CRP) to form a complex cAMP-CRP. cAMP-CRP will bind to the DNA upstream of the promoter and will interact with the RNA polymerase to increase transcription levels.

What is transformation?

The process of importing free DNA into bacterial cells transformation Is the o nly where u have free dna floating around in other cases dna is protected - The genome gets broken up into pieces; plasmidis will get released into the environment; transformation = living cells can pick up this information - transformation: cells are swimming around in environment and when they die they release their genome; the genome usually doesn't stay intact it gets broken yp into pieces and plasmids gets released; free floating dna; living cells can pick up this information; the process of importing free dna in the cells; bacterialcells; in order to do that a cell has to be considered competent

What is the role of regulatory RNAs? How are they used to control gene expression? What level of control would this be considered?

These can be divided into two main types:1. Small RNA (sRNA) molecules, which are encoded by intergenic regions (between genes); 100-200 base pairs in length; come from two locations: come from intergenic spaces AND Within a gene2. cis-antisense RNA (asRNA) molecules, which are transcribed from the DNA strand opposite the mRNA-encoding template strand; comes from within a geneRegulatory RNA molecules typically affect gene expression posttranscriptionally, either by interacting with proteins or by binding to complementary sequences of target transcripts in ways that stimulate or prevent translation

rnaseH

To get red of rna we have RnaseH that degrades RNA that degrades the primer which leaves a gap; the cell can go in and replace the rna with dna; dna poly 1 fills in the gap removes the RNA primers that are bound to the DNA template strands after replication has occurred; a family of non-sequence-specific endonucleases that catalyze the cleavage of RNA via a hydrolytic mechanism; members of the RNase H family can be found in nearly all organisms, from bacteria to archaea to eukaryotes

Is there only one termination ination site?

no

What is a sigma factor? How is the sigma factor involved in transcription

only in bacteria; used to identify the promoter sequence. - Sequence in front of gene; "housekeeping" sigma factor, and may have multiple other sigma factors; Genes you need all the time; things use constantly like glucose metabolism, chaperonesneeded only for initiation of RNA synthesis, not for its elongation

H ow does a cell regulate sigma factors? What is the purpose of sigma factor regulation?

orchestrates the stationary-phase accumulation of sigma S by modulating proteolysis of the factor. ClpXP protease degrades sigma S rapidly during exponential growth. When cells enter stationary phase or experience an environmental stress that slows growth, degradation of sigma S stops. Sigma S levels increase in these situations and trigger the expression of stress survival genesOther sigma factors are controlled by anti-sigma factor proteins that inhibit sigma factor activity. Anti-sigma factor proteins target specific sigma factors and block access to core RNA polymerase. The anti-sigma strategy prevents expression of target genes until they are neededAnti-sigma factors can themselves be neutralized by anti-anti-sigma factors that bind the anti-sigma factor more tightly than sigma factor does. The anti-anti-sigma factor acts as a decoy to release anti-sigma factor from the actual sigma factor. Freed sigma factor can then join core RNA polymerase and direct transcription of target genes

oriC

origin of replication

what are the 3 types of gene transfer in bac

pathogenicity, symbiosis, and fitness islands

What is a promoter/how are they identified/ What is a consensus sequence where are they found in dna

signals the beginning of the genepromoter sequences are characteristically centered at -10 and -35 nt before the start of transcription the sigma factor helps the core enzyme detect the promoterSigma factors generally contain four highly conserved amino acid sequences, called regions. Part of region 2 of the sigma -70 family recognizes -10 sequences, whereas region 4 recognizes the -35 sites similarities among these different promoter DNA sequences define a consensus sequence likely recognized by the sigma factor sequence consists of the most likely base (or bases) at each position of the predicted promoter. Although promoters are double-stranded DNA (dsDNA) sequences, convention is to present the promoter as the single-stranded DNA (ssDNA) sequence of the sense strand (nontemplate strand), which has the same sequence as the RNA product located at -35 has the consensus sequence TTGACA; IN SHINE DALGARNO BOX; The upstream leader RNA contains a purine-rich consensus sequence (5′-AGGAGGU-3′) located four to eight bases upstream of the start codon. This upstream sequence is called the ribosome-binding site or Shine-Dalgarno sequence,

what is the start codon and what does it code for, what do stop codons code for

start: aug --> methionine stop: UAA, UAG, UGA

dna ligase

synthesizes short segments; when the primer has been filled in w dna; dna poly cannot attach with the last nucleotide with the first nucleotide so u end up with a nick; dna ligase (and) attaches these 2 things together

ter

termination seqences

What is a transcription bubble? What type of enzyme is involved? what type of coil is introduced and how is tension relieved

the unwinding of DNA ahead of the moving complex forms a transcription bubbleRNA polymerasepositive supercoils are removed by DNA topoisomerases

What is deoxyadenosine methylase?

thx aleah an enzyme that adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNAHow does SeqA know to bind just after the origin has replicated? The key is DNA methylation. E. coli uses the enzyme deoxyadenosine methylase (Dam) to attach a methyl group to the N-6 position of adenine in the sequence GATC

dnaG (dna primase)

•DNA primase is recruited by each DNA helicase and will prime the leading strand at each fork. -- synthesizes the primer; recruited by the helicase; a primer is a short segment of rna that is completementary to dna; this is key bc dna poly cannot synthesize without a preexisting 3' OH group - so the primase leaves one of these free; now the dna poly can use it to link the next nucleotide -Now the primase is in place the dna poly can read it and polyermate using the 3' OH

what is the holoenzyme

•Holoenzyme= RNA polymerase + sigma factor initiates transcription of a DNA template strand

Understand that proteins may be modified in different ways post translation.

•Not all proteins are immediately functional. •By nature of the genetic code all bacterial polypeptides begin with N-formylmethionine -Remove formyl group -Remove methionine •Other modifications include the addition of small groups such as acetyl or phosphoryl groups. •Some proteins can be cleaved as a method of activation or inactivation.

What is an aporepressor? corepressor? Understand how the tryptophan operon uses this type of repression.

•Regulation of the tryptophan operon •Genes are required for tryptophan SYNTHESIS •Genes encoding biosynthetic enzymes are regulated by repressors, called inactive aporepressors. •These bind the end product of the pathway, which is called the corepressor In contrast, genes encoding biosynthetic enzymes are regulated by repressors (called inactive aporepressors) that must bind the end product of the pathway (for example, tryptophan for the trp operon) to become active repressors. The pathway product that binds the aporepressor is called a corepressor. Binding of the corepressor (end product) to the repressor increases the repressor's affinity for the operator sequence upstream of the target gene or operon. As the concentration of end product, such as an amino acid or nucleotide, increases in the cell beyond what is needed to support growth, the cell will shut the biosynthetic pathway down and not waste energy making a superfluous pathway or compound.The tryptophan biosynthetic pathway in E. coli and repression of the tryptophan operon. The tryptophan biosynthetic enzymes and their encoding genes are shown. TrpR aporepressor (inactive repressor) binds excess tryptophan when intracellular concentration exceeds need. The holorepressor (active repressor) then binds to the trp operator and greatly reduces transcription. Note the long polycistronic message in blue. Repression lowers expression about 100-fold. PRPP = phosphoribosyl pyrophosphate.

What are regulatory proteins? Where do they come from

•Regulatory proteins act bind to regulatory sites on the DNA •Positive regulation= The increased expression of a gene by the binding of an activator protein •Negative Regulation= The decreased expression of a gene due to the binding of a repressor protein

hat is the replisome?

•Replisome- Each replication fork will contain •1 helicase •2 polymerase III •2 sliding clamps •The replisome ensures that the leading and lagging strands are synthesized simultaneously in the 5′-to-3′ direction.

What is the termination utilization substance?

•Terminator sequences are bound by a terminus utilization substance (Tus). -What u dont want to happen is one replication gets way far ahead of the other one and start replicating into the others territory; in these ter sequences there are proteins thtat bind called tus; they bind to particular sequences and act to trap the fork tthats going in the opposite direction; one rep fork going counter-clockwise so it can sequence thru all of these without the problem but if it encounters these on the other fork it stops working; so it acts as a roadblock

What happens to a cell if the F - factor plasmid integrates into the chromosome?

•The F-factor plasmid can integrate into the chromosome. •The cell is now designated Hfr, or high-frequency recombination strain.

How does the AraC regulator both activate and repress transcription? What catabolic pathway is AraC regulating?

•The arabinose operon encodes genes required for arabinose metabolism. •AraC protein regulates this operon and can act as both an activator and a repressor. - If arabinose is not present and the genes in the AraC operon are necessary to break down arabinose; I want this repressed; I want to repress transcription; bc theres no arabinose the wayt his dimer comes together so the binding sites are 180 degrees away from each other; the regions that these DNA binding sites between 2 are the yellow or the light green; but theyre far away so I cant bind to both at t he same time; when I binid to one of them the only other region the binding site can interact with is further downstream which causes thte DNA to bend in this direction; therefore, im blocking the promoter; rna poly cant transcribe Iif my dna binding sites are right next to e ach other I have an opposite situation; they interact with sequential re cognition sequences; I end up acvtivating; increase level s of transcription AraC protein regulates this operon and can act as both an activator and a repressor.Arabinose operon regulated by AraC; encodes for genes necessary for arabinose metabolism; if you don't have arabinose, don't want to transcribe genes in that operon: same as lac; one protein, AraC itself can act as activator or repressor; only one protein compared to two in lac; presence of arabinose determines if it acts as repressor or activatorRegulatory protein functions as a dimer; C terminal DNA binding domain: where binds to DNA regulatory sequence; flexible linker: DNA binding domain can change orientation; N terminal dimerization domain: where two subunits come together to dimerize; occupation of arabinose binding site determines how dimer comes togetherBlock CRP site when DNA binds around; because of way two dimers oriented will block promoter, no transcription of operon; when arabinose absentArabinose present, bind to araI1 and araI2 (regulatory sequences and so is RP), DNA binding domains side by side, no longer blocking promoter, opens up to be transcribed, actives transcription

Initiation

•The origin of replication is a conserved 250 bp sequence. •Recognized and bound by DnaA •Blocked by SeqA •The binding of the origin proteins results in an opening of the DNA helix which is necessary for replication to begin. •DnaB (helicase) will be loaded onto the exposed replication forms. •Clamp loader then loads the DNA polymerase III along with the sliding clamp onto the DNA strand ADD MORE TO THIS

What is transduction

•Transduction is the process in which bacteriophages carry host DNA from one cell to another. 1. Generalized transduction: can transfer any gene from a donor to a recipient cell 2. Specialized transduction: can transfer only a few closely linked genes between cells

what is universal genetic code? what is a codon and where is it located what is an anticodon and wheres it located

•Universal genetic code •Codons •Redundancy •Stop Codons - information stores in the form of nucleic acids An anticodon is at the bottom, middle, loop of all tRNA molecules; triplet that basepairs with codons in mRNA