Micro Exam 3

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Gene: Functional unit of genetic material. Includes the information encoded within DNA that specifies the sequence of AA of a protein as well as the regulatory sequences that control its expression
DNA: a macromolecule containing all of the information for life processes.
RNA: Single-stranded but the RNAs fold back on themselves and form complicated secondary structures due to intrastrand base pairing.
Transposable Elements: Includes:
Transposons-contain genes
Insertion elements-no functional genes
Conjugative transposons-
Length of each Base Pair: 0.34 nm (and each turn of the helix contains 10 bps; so 1kb of DNA has 100 turns and is 0.34 micrometers long)
Inverted Repeats: Short sequences that are repeated in an inverse orientation relative to each other. Because they are inverted, they can form stem-loop structures (cruciforms), which are often important for protein interactions.
Thermal Melting of DNA: Gives a measure of the G + C content.
Hybridization: The artificial construction of ds Nucleic Acid (DNA:DNA, DNA:RNA or RNA:RNA) Typically done in the lab to identify particular DNA or RNA sequences.
Negative supercoiling: occurs when the DNA is twisted about its axis in the opposite direction from that of he right-handed double helix. (The predominantly found form)
DNA gyrase: ONLY in Bacteria and Archaea. Type II Isomerase. Introduces negative supercoils by making double-strand breaks in the DNA and twisting the DNA in the process. After the DNA is supercoiled, the break in the DNA backbone is repaired by an enzyme called DNA Ligase.
Topoisomerase I: catalyzes the opposite reaction from DNA gyrase and relaxes the negatively supercoiled DNA. This is accomplished by Topo I making a single strand break (nick) in the DNA backbone. After the DNA relaxes, the nick is sealed.
(To prevent the entire bacterial chromosome from becoming relaxed everytime a nick is made, the chromosome contains supercoiled domains)
Prokaryotic DNA is:: -supercoiled
-circular
-naked (mostly devoid of bound protein)
Eukaryotic DNA is:: -Linear
-Compacted by coiling around positively charged histone proteins, forming nucleosomes and then chromatin.
(Histones imparte negative supercoiling)
Inhibit the action of DNA gyrase:: Quinolones such as nalidixic acid, fluoroquinolones such as ciprofloxacin, and novobiocin.
Novobiocin is also effective against DNA gyrase in Archaea.
Primase: An RNA-polymerizing enzyme that participates in primer synthesis by synthesizing a short stretch of RNA. There is a 3'OH group at the growing end of this RNA primer where DNA pol can act on.
(The primer must eventually be removed.)
DNA Polymerases: -enzymes that catalyze the synthesis of DNA from a DNA template.
-ALL DNA pol synthesize new DNA in the 5'->3' direction.
-NO known DNA pol can begin a new chain...they must add to a pre-existing 3'OH group. Therefore, there must be a primer present.
-DNA pol can only recognize DEOXYribonucleotide triphosphates.
DNA replication occurs in what direction?: DNA replication always proceeds in the 5'->3' direction (template is actually 3'->5')
The 5'-P of the incoming nucleotide being attached to the 3'-OH of the previously added nucleotide.
Origin of Replication: is a specific sequence of about 300bp. Here the DNA is made single-stranded to allow it to be replicated.
Specific proteins recognize the Ori to initiate DNA replication in Prokaryotes.
A Euk chromosome has many Ori while a Prok has ONLY one.
Events at Replication Fork: 1. ATP-dependent Helicase unwinds dsDNA
2. ss binding proteins bind to ssDNA and prevent it from becoming ds again
3. Primase is an RNA pol that makes an RNA primer that allows DNA pol to initate DNA replication.
4. DNA pol III is the pol that adds most of the nucleotides to the growing DNA strand.
5. DNA pol I uses long DNA strands as primers and begins adding deoxyribonucleotides in the place of RNA. It gets rid of the RNA primer.
6. DNA pol I can remove the RNA primer because it has 5'->3' exonuclease activity, DNA pol III does not.
7. DNA ligase joins the new DNA strands on the lagging strand.
DNA Polymerase III: has 5'->3' DNA pol activity and 3'->5' exonuclease activity.
DNA Polymerase I: can synthesize DNA, however, at the same time it is adding nucleotides to to the 3'OH, it has a 5'->3' exonuclease activity that removes the RNA primer from in front of it. When the primer has been removed and replaced with DNA, DNA pol I is then released.
Helicase: Is attached to the CM and pulls DNA through it and unwinding dsDNA for replication.
Replisome: A complex enzyme (has helicases, primase, two DNA pol III molecules, and other proteins) associated with CM that pulls DNA through (like helicase).
The two DNA strands are being syn by a duplex of DNA pol. This is made possible by a "looping" of the lagging strand.
Proofreading: Occurs because of unstable base pairing.
DNA pol III : 3'->5' exonuclease activity.
DNA pol I : 5'->3' exonuclease activity
DNA methylases: enzymes that recognize and methylate palindromic sequences.
Restriction endonucleases or restriction enzymes: will cut "unmethylated" DNA. Therefore, foreign DNA that is unmethylated is destroyed while its own DNA is replicated.
Type I and III enzymes carry methylation and ATP-dependent restriction (cleavage) activities in the same protein.
Type I bind recognition sequence on DNA and cleave at random spots.
Type II cleaves specific sequences, and have a separte methylase that modifies the same recognition sequence.
Problems with replicating the ends of linear DNA, how are these ends replicated?: 1. circularize DNA using sticky ends
2. utilize an OH group of a special protein
3. Hairpin turns at the end of ssDNA
4. Telomerase adds extra pieces of DNa to ends of EUKARYOTIC DNA.
There are probably other solutions also.
Nucleic Acid synthesis can be described by five rules:: 1. Both DNA and RNA chains are produced in cells by the copying of a preexisting DNA strand according to the rules of Watson-Crick basepairing.
2. Nucleic acid strand growth is in one direction 5'->3'
3. special enzymes called polymerases make DNA or RNA
4.Duplex DNA synthesis requires a special growing fork
5. RNA processing is a key event in making functional RNA.
Start Codon: AUG
Stop Codons: UAA, UGA, and UAG
Staphylococcus: -Gram + Cocci in clusters.
-about 1 micrometer in dia.
-tolerate high salt (up to 15%)
-tolerate low water potentials
-non-motile
-nonsporeforming
-Facultative anaerobe (heterofermenter)
-Catalase positive (distinguish from Streps, which are catalase negative)
Staphylococcus epidermidis: forms small white colonies, nonpathogenic microorganism commonly found on skin.
Leads to body odor.
Staphylococcus aureus: forms large yellow colonies, nonpathogen AND an opportuistic pathogen. Can be a pathogen through breaks in skin.
Additional S. aureus characteristics:: -Ferments mannitol (distinguishes S. aureus from S. epidermidis)
-Coagulase positive-causes blood clotting, shields organism from phagocytes. (distinguishes S. aureus from S. epidermidis)
-causes a variety of suppurative (pus-forming) infections and toxinoses in humans.
--Acne and Boils
--Styes-localized inflammatory of sebaceous glands of eyes
--pneumonia
--mastitis
--urinary tract infections
--noocomial infections
--food poisoning
--toxic shock syndrome-by creating a super antigen
Mannitol: Mannitol is NOT converted to glucose. Instead, mannitol is oxidized to fructose-6-P.
mannitol->mannitol-6-P (by the PEP system.
mannitol-6-P + NAD -> NADH2 + frc-6-P (by mannitol-6-P DH)
frc-6-P enters glycolysis -> pyruvate.
Potential virulence factors of S. aureus infections:: 1. surface proteins promote colonization of host tissue.
2. invasins promote spread in tissues: leukocidin, kinases, hyaluronidase, streptokinase (lyse fibrin and causes dissolution of fibrin clots, may aid in spreading.
3. anti-phagocytic factors: carbohydrate microcapsule, protein A, perhaps coagulase.
4. enhanced survival in phagocytes; catalase
5. immunological disguises: protein A, coagulase
6.Host-damaging toxins: hemolysins, leukotoxin, leukocidin (alpha-toxin, beta-toxin, leukocidin)
7. provoke disease symptoms (enterotoxin, exfoliative toxin, superantigens)
8. Inherent and acquired antibiotic resistance: MRSA
alpha-toxin: (a-hemolysin); pokes holes in membranes
beta-toxin: sphingomelinase; destroys membranes rich in the lipid sphingomelin
Leukocidin: multicomponent toxin that forms pores in leukocyte (white cells or immune cells) membranes, somewhat hemolytic but less lytic and alpha-hemolysin.
Enterotoxin: (acts on intestines) causes vomiting when ingested - a leading cause of food poisoning.
Exfoliative toxin: causes scalded skin syndrome in neonates - wide spread blistering and loss of epidermis.
Superantigens: stimulate T cells non-specifically to release large amounts of cytokines
Endonucleotyic Enzymes: identify misinserted nucleotides. Cut the DNA near the misincorporated nucleotide and remove it completely.
Pol I repairs the gap and DNA ligase seals it.
mRNA: carries the genetic information needed to encode proteins.
rRNA: serves a functional and structural role by being a major component of ribosomes.
tRNA: Plays a functional role in protein synthesis (translation) by being the component that carries AAs.
RNA synthesis:: -RNA pol does not require a primer
-proceeds in 5'->3' direction
-most of the time requires DNA as a template.
Bacterial and Arcaeal RNA polymerase: -Bacteria and Archaea have a SINGLE type of RNA pol to synthesize mRNA, rRNA, and tRNA.
Core RNA pol has beta, beta-prime, and two alpha subunits
Holoenzyme has beta, beta-prime, alpha, and sigma subunits. (sigma falls off after RNA syn begins.)
Eukaryotic RNA polymerase: Eukaryotes have 3 different types of RNA pol, RNA Pol I, RNA Pol II, and RNA Pol III.
Sigma: role is to recognize the appropriate site of the DNA of RNA pol for the initiation of RNA syn. Sigma is involved only in the RNA Pol/DNA complex.
The sigma factor is the part of the RNA Pol that recognizes the promoter sequence.
Promoter: The sequence of the DNA that specifies where RNA Pol binds and to which strand of the DNA.
Transcription Terminators: Specific bases in the DNA that stop transcription of mRNA.
Consensus Sequence: represents the bases that are usually present in the promoter in each position after comparing many different sequences.
Promoters with sequences closer to the consensus sequence are strong promoters.
Intrinsic transcriptional terminators (Rho-independent): Sequences such as regions of DNA that are GC rich and are inverted repeats in Bacteria. The inverted repeats in RNA allow the stem loop structure to form in the RNA. If this stem loop is followed by a run of "U"s it causes RNA Pol to fall off the RNA/DNA interface.
Rho-dependent Transcriptional Termination in BACTERIA (not in Eukaryotes): Rho binds the RNA being transcribed, NOT the DNA, and begins moving down the RNA towards the RNA pol/DNA complex. At the 3' end of the Rho dependent gene, RNA Pol pauses at a Rho-dependent termination site and then RNA pol falls off the DNA.
RNA polymerase Inhibitors: Rifamycins-inhibit the beta subunit of RNA Pol
Streptovaricins-similar to rifamycins but bind to a different site on the beta-subunit.
Amanitin-fungal compount that inhibits eukaryotic RNA Pol II. USED to be used for treating certain viral diseases.
Actinomycin-interacts with G:C base pairs and blocks transcriptional elongation.
Types of Eukaryotic RNA Polymerases: RNA Pol I: transcribes most types of rRNA
RNA Pol II:transcribes all the mRNA
RNA Pol III:transcribes tRNA (and one type of rRNA)
Transcription factors must be present on promoters before Pol II can bind.
MINIMAL RNA Pol II promoters:
-TATA box
-an initiator element near the transcriptional start site
-variety of other promoter-specific sequences that bind specific transcription factors
Transcription Factors: Either postion RNA Pol II on the transcriptional start site or prevent RNA Pol II from binding.
Trancriptional Termination in Eukaryotes: Trancriptional termination is signaled by a specific sequence. Most, but not all, eukaryotic mRNAs are terminated at the 3' end with a poly (A) tract.
Poly(A) polymerase: Adds many A's on to 3' end of primary mRNA

A base sequence -AAUAAA- located 10-25 bases upstream from the poly(A) site is required for poly(A) addition.
The length of poly(A) tail can be from 20-200 nucleotides.
RNA editing in Eukaryotes: 1. 5' end of eukaryotic RNA is modified by the addition of a 7-methyl-Guanine cap
2. Newly synthesized mRNAs must have their introns removed by RNA splicing
3. Addition of poly(A) tail on 3' end.

All occur in nucleus before mRNA is exported to cytoplasm.
tRNA characteristics: -short molecules (75-100 bases) with an overall cloverleaf-like structure.
-contain a site for AA attachments
-has an Anticodon that base pairs with a codon of the mRNA.

Proper selection of the AAs is determined by positioning the tRNA molecules on the mRNA.
tRNA structure: 1.anticodon loop
2.TYC loop contains several modified bases including a psuedouracil (Y)
3.D loop contains modified bases (dihydrouracil, D)
4.CCA acceptor terminus is invariant and serves as the attachment site for AAs
aminoacyl-tRNA synthetase: charge or acylate the ends of the tRNA molecules with tRNA-specific AAs

There is at least one and usually only one aminoacyl synthetase for each AA

Charging of the tRNA with an AA requires ATP hydrolysis to AMP + 2Pi
Prokaryotic Ribosomes: 30S subunit= 16S rRNA, 21 proteins and has the mRNA threaded through it.
50S subunit= 5S and 23S rRNAs, 34 proteins and it contains the tRNA binding sites.

30S + 50S = 70S ribosome

Contains a E-site, P-site, and A-site which are occupied by a.a.-tRNAs
Eukaryotic Ribosomes: 40S subunit= 18S rRNA and approx. 30 proteins
60S subunit= 28S rRNA, 5.8S rRNA, and 5S rRNA and 50 proteins.

40S + 60S = 80S ribosome
Translation Initiation: 1. initiation begins with h-bonding of f-met-tRNA to start codon (AUG)
2. GTP binds to a specific site on the 30S subunit
3. Final ribosome assembly invovles the association of the 50S subunit, GTP hydrolysis and release of the IFs
4. At some point, the formyl group is removed.
Traslation Elongation: 1. aa-tRNA first binds to a protein complex called elongation factor Tu and GTP
2. EF-Tu places the aa-tRNA in the A site
3. GTP is hydrolyzed to GDP + Pi
4. EF-Tu-GDP is released and recharged with GTP.
5. Formation of the peptide bond is accomplished by the peptidyl-transferase activity of the 23S subunit.
Translation Translocation: 1. Ribosome moves down the mRNA three nt and moves the free tRNA to the E-site
2. Translocation is catalyzed by elongation factor G (EF-G) and requires GTP hydrolysis
3. translocation leaves the A-site open for the next incoming aa-tRNA
4. in this process, all ribosomal elongation factors are released
Translation Termination: at stop codon, no tRNAs bind but release factors cleave the polypeptide from the complex.
Internal Tranlational Initiation: Bacterial 30S subunit can bind to internal shine-delgarno sequences upstream of internal open reading frames and initiate translation.
Translational Reinitiation: The small subunit of rRNA can remain attached to mRNA after the stop codon and continue scanning downstream for the next AUG start codon.
Polysome: a complex of mRNA and multiple ribosomes.
Multiple ribosomes can simultaneously translate mRNAs
Chaperonins: -ATP dependent
-Chaperonins do not contribute conformational information to the folding process.
-allow proper protein folding
EUKARYOTIC transcriptional termination: transcriptional termination is signaled by a specific sequence AATAAA or AAAUAA.

Transcription terminates 0.5-2.0kb downstream (3') of the AAAUAA or AAAUUA sequence in mRNA

Poly(A) polymerase is thought then to:
1. cleave the RNA 10-30 nucleotides downstream of AAAUAA
2. add 20-250 residues to the 3'end.
Polycistronic mRNAs: many protein-encoding genes are transcribed as one long mRNA molecule

not generally found in Eukaryotes.
Operon: a complete unit of gene expression in PROKARYOTES. Often related enzymes are clustered together so that their expression is regulated by a single promoter which contains a regulatory element (operator) that can enhance or prevent initiation of RNA transcription.
Inducer: binds to the repressor protein causing it to fall off the DNA

Regulates gene expression in PROKARYOTIC mRNA!
Corepressor: binds to the repressor protein causing it to bind to the DNA. The repressor cannot bind without the inducer.

Regulates gene expression in PROKARYOTIC mRNA!
ribozymes: self-splicing introns in some bacteria
Translation Initiation in EUKARYOTES: the 40S subunit recognizes and binds to the 5'end of the mRNA via the 7-methyl-guanine cap.
This small subunit then scans down the mRNA until the FIRST AUG codon is found.
Initiation factor 4 (eIF4) is critical in finding the AUG.
The large subunit joins the complex after the 40S, met-tRNA, and eIF4 find the AUG
Antibiotics that affect protein synthesis: Streptomycin: inhibits initiation. Also, negatively affects euk mitochondria.

((Puromycin, chloramphenicol, cycloheximide and tetracyline inhibit elongation.))

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