Microbiology exam 3

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Microbiology exam 3
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  1. repetitive process of replication, transcription, and translation
    build polymers of nucleotides or amino acids
  2. 3 major steps of replication, transcription, and translation (RTT)
    • initiation
    • elongation
    • termination
  3. when RNA polymerase binds to promoter, it forms a
    closed complex
  4. RNA polymerases binds promoter, 10 and 35 bases upstream of start site
  5. step in RTT, binds polymerizing machine, first monomer to template
    initiation
  6. 3 structures involved in initiation
    • DNA polymerase
    • RNA polymerase
    • ribosomes
  7. step in RTT that reads template and adds next monomer
    elongation
  8. 3 structures involved in elongation
    • DNA
    • RNA
    • protein
  9. step in RTT that release machine and complete product
    termination
  10. RNA polymerase is a large molecular machine
  11. this enzyme has 4 protein units in one complex
    RNA polymerase
  12. function of RNA polymerase
    binds DNA and reads sequence
  13. Where does RNA polymerase bind to DNA
    sigma factor
  14. 3 functions of sigma factor
    • specificity
    • guides RNA polymerase to DNA
    • promoter
  15. core + sigma
    holoenzyme
  16. 2 characters of sigma factors
    • each for subset of genes
    • contain diff promoters
  17. function of RpoD sigma70 genes
    housekeeping
  18. most genes from sigma factors are
    RpoD sigma 70
  19. RpoD, sigma 32 functions when
    activate when the cell is stressed by heat
  20. heat shock response proteins
    • chaperones
    • proteases
  21. sigma factors binds core RNA polymerase which forms
    RNA polymerase holoenzyme
  22. mRNA func
    encodes protein
  23. rRNA func
    synthesizes protein
  24. tRNA func
    shuttles amino acid
  25. sRNA
    controls transcript and lation
  26. tmRNA func
    free ribo stuck on damaged mRNA
  27. catalytic RNA func
    carries out enzymatic rxns
  28. genetic code consists of nucleotide triplets
    codons
  29. 64 possible codons
    61 specify AA (w/ start codon)
    3 stop codons
  30. genetic codes are degenerate or redundant
  31. multiple codons can encode same amino acid
  32. given DNA sequence in transcript and lation can encode 2 mRNAs
  33. in protein synthesis, transcript and lation are directional and one is used as a template
  34. in protein synthesis, a set of tRNAs bind individual amino acids
  35. 2 characters of tRNA
    • have specific shape
    • have 3 base anticodon (base pair to codons in mRNA)
  36. proteins add AA to tRNA, enzyme used is
    aminoacyl-tRNA transferase
  37. when proteins add AA to tRNA, what kind of E is required and what does it get reduced to
    • ATP
    • AMP
  38. function of aminoacyl tRNA synthetases
    enzyme that matches amino acid to correct tRNA
  39. DNA and proteins can be labeled separately by using radioactive S and P, which letter goes to which
    • P for DNA
    • S for Proteins
  40. 2 types of gene transfer
    • vertical
    • horizontal transmission
  41. vertical transmission is the transfer from
    parent to child
  42. horizontal transmission is the transfer of
    small pieces of DNA from one cell to another
  43. produces a functional RNA which usually encodes a protein
    structural gene
  44. regulates the expression of structural gene
    DNA control sequence
  45. DNA control seq does not encode an RNA
  46. dna polymer is a polymer made up of nucleotides
  47. each nucleotide of DNA is made up of 3 parts
    • nitrogenous bases
    • deoxyribose sugar
    • phosphate
  48. the 2 nitrogenous bases types
    • purine
    • pyrimidine
  49. 2 purine
    • adenine
    • guanine
  50. 2 pyrimidine
    • cytosine
    • thymine
  51. nucleotides are connected to each other by
    5-3' phosphodiester bonds
  52. 4 components to the nucleic acid structure
    • sugar (deoxyribose)
    • 3 phosphate groups on 5' C
    • phosphate links to 3' OH of the next base
    • base of DNA attached to sugar
  53. order of bases on
    DNA
    RNA
    • Adenine, guanine, cytosine, thymine
    • adenine, guanine, cytosine, uracil
  54. 3 ways RNA is different from DNA
    • single stranded
    • contains ribose sugar
    • uracil replaces thymine
  55. complimentary sequence of
    5'-ATG-CTT-AAG-3'
    5'-CTT-AAG-CAT-3'
  56. DNA is packed to fit the cell
  57. ex of how DNA is packed to fit the cell
    nucleoid of e.coli is composed of circles of dsDNA 1500x the size of the cell
  58. how is nucleoid of E.coli able to be packed into the cell 2
    • multiple loops are held by anchoring proteins
    • each look has coiled DNA
  59. unsupercoiled DNA=
    1 wind for 10 BP
  60. 10bp=3.4nm
  61. supercoiling compacts DNA
  62. this kind of winding is more frequently, overwinding occurs
    positive supercoiling
  63. this kind of winding has winding that is less frequently, underwinding occurs
    negative supercoils
  64. this kind of coiling twists DNA
    supercoils
  65. what helps to regulate supercoils
    topoisomerases
  66. 3 kinds of topoisomerases
    • type I
    • type II
    • archael
  67. this kind of topo relieves torsional stress caused by supercoils
    type I
  68. this kind of topo has DNA gyrase and introduce negative supercoils
    type II
  69. this kind of topo reverse DNA gyrase and introduce positive supercoils
    archael
  70. DNA replication
    (semiconservative replication)
    copies info from one strand to a new complimentary strand
  71. why is DNA known as semiconservative replication
    due to accuracy check
  72. how to DNA replication start off 2
    • melt double stranded DNA
    • polymerize new strand complementary to each melted single strand
  73. 6 major proteins involved in DNA replication
    • DNA-A
    • DNA-B
    • DNA primase
    • DNA pol III
    • DNA pol I
    • DNA gyrase
  74. DNA-A function
    initiator protein
  75. DNA-B
    helicase
  76. DNA primase function
    synthesis of DNA primer
  77. DNA pol III function
    major replication enzyme
  78. DNA pol I function
    replaces RNA primer with DNA
  79. DNA gyrase function
    relives DNA supercoiling
  80. where does replication begin
    at oriC
  81. When DNA melts at oriC, what 2 things are occuring
    • DNA-A activates
    • Seq-A represses
  82. when replication begins, what 2 things are happening
    • DNA melts at oriC
    • polymerization follows melting around the chromosomes
  83. What helps to melt DNA for replication
    DNA helicase
  84. How does helicase melt DNA 2
    • loader places helicases at each end of the origin
    • one helicase moves in each direction to copy each genome
  85. when helicase recruits primase, what 2 things occur
    • primase will begin the replication
    • RNA primer forms 3' OH for DNA to attach
  86. first cells were thought to use RNA not DNA
  87. when primer recruits clamp loader to each strand, what happens
    clamp binds DNA polymerase III to strand
  88. polymerase replicates from 5 -->3 on each strand
  89. energy for polymerization for DNA replication comes from where
    phosphate groups on added bases
  90. when polymerization occurs, it must add a new base to 3 OH of a chain, while new nucleic acid grow to extend 3 end
  91. each replication fork has two strands
  92. at each fork in replication there is a steadygrowth of new leading strand (follows helicase)
    and a single DNA strand for half of chromosomes
  93. 2 occurs while the lagging strand grows during replication
    • polymerase continues to the previous primer
    • clamp loader places primase on new site
  94. when the lagging strand grows, it is done by the okazaki fragment
    DNA has 1000 present in the okazaki fragments
  95. RNAse H function
    removes primers
  96. there is 1 primer for each leading strand in DNA rep
    There are many primers of lagging strand in DNA rep
  97. there is one primer per okazaki fragment
  98. the gaps of the DNA rep are filled by ____ and the nicks are sealed by _____
    • polymerase I
    • ligase
  99. when both forks move to ter sites 3 things occur
    • movement is simulatenous
    • move opp ways until they meet again at terminus
    • replisomes are actually stationary
  100. DNA gets threaded through replisomes
  101. are extrachromosomal pieces of DNA
    plamsids
  102. low copy number plasmids 2
    • 1 or 2 copies per cell
    • segregate similarly to chromosome
  103. high copy number plasmids 3
    • up to 50 copies per cell
    • divide continuously
    • random segregation to daughter cells
  104. 2 ways plasmid replication can occur
    • bidirectional replication
    • unidirectional replication
  105. plasmid bidirectional replication is similar to chromosomal replication
  106. this type of plasmid replication is used by many bacteriophage viruses
    unidirectional
  107. how unidirectional plasmid replication work
    • starts at nick bound by RepA protein
    • provides 3 OH for rep
    • helicase moves around plasmid repeatedly
    • complementary strand synthesized
  108. plasmid genes are advantageous under special conditions
  109. plasmid genes can perform 5 functions amongst others examples
    • antibiotic resistance
    • resistance to toxic metals
    • metabolize rare food sources
    • virulence genes to allow pathogens
    • allow symbiosis
  110. 2 ways DNA can be manipulated
    • restriction enzymes cut DNA at specific sites
    • PCR uses short oligonucleotides
  111. restriction enzymes are used to
    normally protect bacteria from viral DNA
  112. when the restriction enzyme cuts the DNA, it reveals location of specific sequence
  113. PCR use of short oligonucleotides cause these 3 to occur
    • primers binding to complementary sites
    • archael enzymes syn DNA
    • amplifies segment of DNA
  114. restriction endonucleases
    cleaves DNA at specific recognition sites which are usually 4-6 bp and palindromes
  115. when restriction endonucleases occurs, it may generate blunt or staggered ends
  116. agarose gel electrophoresis function
    can be used to analyze the DNA fragments obtained by treatment with restriction enzymes
  117. dna amplification
    polymerase chain reaction
  118. 4 events in PCR
    • denaturation
    • annealing
    • extension
    • repetition
  119. PCR uses heat stable DNA polymerase
  120. whole genome sequence 3 characteristics
    • break genome into 1,000's of pieces
    • determine sequence of many short pieces
    • computer determine sequence overlap to recreate entire genome sequences
  121. most commonly used DNA sequencing method relies on the
    sanger dideoxy strategy
  122. what is the sanger dideoxynucleotide strategy
    incorporation of 2',3'-dideoxynucleotide into a growing chain preventing further elongation
  123. bacterial chromosome is packed in a series of protein bound loops collectively called
    nucleoid
  124. are enzymes that super coil DNA
    topoisomerases
  125. DNA replication is divided into 3 phases
    • initiation
    • elongation
    • termination
  126. DNA replication:
    initiation occurs
    elongation occurs
    termination occurs
    • at the origin (oriC)
    • at the replication fork
    • at the terminus
  127. each phase of replication requires a # of different proteins
  128. plasmids are autonomously replicating extrachromosomal DNA elements
  129. plasmids benefit the host under certain conditions
  130. 4 ways DNA can be analyzed
    • restriction enzymes
    • gel electrophoresis
    • PCR
    • DNA sequencing
  131. polymerase unwinds DNA at promoter known as
    open complex
  132. once polymerase begins to unwind, what happens to the sigma factor
    it is released
  133. which part of the RNA polymerase recognizes the -10 region of a promoter
    region 2 of sigma factor
  134. core polymerase adds RNA to 3' end
  135. energy for base addition comes from the base
    mRNA (n) + NTP = mRNA (n+1) + diphosphate
  136. added base in transcription elongation when polymerase adds RNA is
    complementary to template strand
  137. mRNA in transcription elongation of RNA has the same sequence as sense strand
  138. For transcription termination of RNA is depends on what to help the termination
    rho-dependent
  139. When termination of RNA transcript occurs, what happens
    RNA polymerase slows at pause site (has a GC-rich sequence)
  140. how does Rho cause termination of RNA transcription
    • Rho factor binds to mRNA
    • slides along the mRNA to polymerase
    • Breaks off polymerase and mRNA off of DNA
  141. 3 characteristics of Rho independent termination
    • DNA seq containing intrinsic terminators
    • inverted repeats followed by runs of Us
    • stem loop structure in RNA
  142. what 4 subunits are present during the closed complex during transcription
    alpha, beta, sigma, gamma
  143. transcription and translation are directional
    • given DNA seq can encode 2 mRNAs
    • usually only one strand is used as template
  144. translation of RNA to protein
    initiation
    initiation factors bind ribosome to start codon
  145. translation of RNA to protein
    elongation
    EF- Tu, EF-G bring GTP energy (polymerization)
  146. polymerization
    movement of ribosome along mRNA
  147. translation of RNA to protein
    termination
    release factors undock ribosome from mRNA
  148. 3 characteristics of tRNA protein synthesis
    • bind to individual amino acids
    • have a specific shape
    • have a 3 base anticodon
  149. proteins adding amino acid to tRNA is called
    • aminoacyl-tRNA transferase
    • requires E of ATP--> AMP
  150. wobble position of anticodon during protein synthesis of tRNA allows
    certain tRNAs to read more than 1 codon
  151. aminoacyl-tRNA synthetases function
    an enzyme that matches amino acid to correct tRNA
  152. protein polymerase
    ribosome
  153. structure of ribosome 3
    • 52 proteins
    • 3rRNAs
    • 2 subunits
  154. ribosome can bind these 2
    • 1 mRNA
    • 3 tRNAs
  155. rDNA operon is transcribed as a single molecule
  156. replication of tRNA has multiple copies of the operon
  157. peptidyltransferase
    23S rRNA of 50S subunit catalyzes peptide bond formation
  158. the 70S ribosome harbors 3 binding sites for tRNA
    • A site
    • P site
    • E site
  159. This site binds incoming aminoacyl-tRNA
    acceptor site
  160. this site harbors the tRNA with the growing polypeptide chain
    peptidyl tRNA site
  161. this site binds a tRNA recently stripped of its poplypep
    exit site
  162. alignment of bacterial structural gene with its mRNA transcript
  163. this antibiotic that affect translation inhibits 70S ribosome formation
    streptomycin
  164. this antibiotic that affect translation inhibits aminoacyl-tRNA binding to the A site
    tetracycline
  165. this antibiotic that affect translation inhibits peptidyltransferase
    chloramphenicol
  166. this antibiotic that affect translation triggers peptidyltransferase prematurely
    puromycin
  167. this antibiotic that affect translation causes abortive translocation
    abortive
  168. this antibiotic that affect translation prevents translocation
    fusidic acid
  169. enzymes modify translated proteins
  170. fMet removed from N-terminus in protein modification
  171. 3 groups added to AA in protein modification
    • phosphoryl
    • methyl
    • adenylate
  172. a protein can be cleaved or refolded by helping enzymes in protein modification
  173. protein structure is determined by
    • AA seq
    • chaperones
  174. 2 kinds of chaperones involved in protein folding
    • GroEl-GroES complex
    • DNAK protein
  175. function of Gro-EL GroES complex
    refolds denatured proteins
  176. how are proteins refolded
    by the use of ATP
  177. barrel shaped proteins that are apart of the GroES complex function
    helps the pro to be refolded fit into the center
  178. all cells constantly rebuild themselves
    proteins are degraded when not needed
  179. proteins survive for minutes to days
    sequence, shape, function determine half life
  180. proteases cut proteins
    cut at specific amino acid sequences
  181. proteasomes degrade proteins and have a barel shape
  182. eukaryotes add signal to proteins, what does the tag do
    ubiquitin tag causes degradation
  183. protein traffic: many bac proteins reside in cytoplasm, others are targeted to other sites, and have signal sequence
  184. 4 targets proteins can be targeted to
    • PM
    • periplasm
    • gram- outside mem
    • secreted outside bacterium
  185. signal sequence
    target protein for transport
  186. 3 characteristics of protein secretion: signal sequence
    • n-terminal AA
    • bound by SRP
    • target ribosomes to plasma membrane
  187. for signal sequencing, they target ribosomes to plasma membrane. what two ways can the proteins be inserted into their final destination
    • some proteins enter plasma mem directly
    • others require secYEG translocon
  188. post translational secretion: SecB function
    protects unfolded polypeptides
  189. post translational secretion: SecA function
    uses ATP to push peptide through SecYEG
  190. post translational secretion: Signal peptide function
    cleaves N-terminal sequence
  191. post translational secretion: TAT system
    exports unfolded proteins using PMF
  192. gram- bactria need to export proteins completely out of the cell
  193. six elegant secretion types have evolved, labeled type I-VI, what are two of their functions
    • some deliver te exported proteins to other dedicated transport proteins in the periplasm
    • others provide nonstop service
  194. genome provides raw DNA sequence
  195. what DNA sequences encode proteins
    open reading frames
  196. what DNA sequences control expression
    promoters
  197. what proteins perform specific function
    biochemical analysis of protein function
  198. genes duplicated via appearance of new species
    orthologs
  199. genes duplicated within a species
    paralogs
  200. in orthologs, identical function in diff organism
  201. in paralogs, they perform slightly diff tasks in cell and can develop new capabilities
  202. transcription is carried out by
    a complex enzyme called RNA polymerase
  203. function of sigma factor
    recognizes the promoter and the core polymerase elongate the RNA strand until a termination signal is reached (may or may not depend on Rho protein)
  204. there are 6 diff classes of RNA
  205. tRNA shuttle amino acid to ribosomes
  206. 3 phases of RNA translation
    • initiation
    • elongation
    • termination
  207. translation RNA initiation
    ribosomal subunits come together
  208. translation RNA elongation
    AA are polymerized
  209. translation RNA termination
    completed protein is released
  210. cell possess diff protein degrading machines
  211. bioinformatics
    use of computer resources to mine and compare genome of organisms
  212. during DNA replication, each nucleotide adds on to
    hydroxyl group on the 3C of the sugar
  213. restriction enzymes do not cut bac DNA that produce them because
    the DNA is protected by site-specific methylating enzymes
  214. if the DAM enzyme is inhibited in E.coli, then the next generation time
    will increase due to more persistent seqA binding
  215. why is the shuttle vector named so
    contains a replication origin compatible with Ecoli and a second origin that will allow the plasmid to replicate in a eukaryote or archae
  216. organisms differ in their gene size
  217. a gene is
    a string of nucleotides that can be used as a template to produce a functional RNA
  218. bactria, archae, and eukaryotes all have what kind of genomes
    double stranded DNA genomes
  219. what is present in DNa sequencing reaction but not in vivo
    dideoxynucleotides
  220. only RNA contains a hydroxyl group on the 2' carbon of ribsose
  221. At 72 degree stage
    thermostable DNA poly syn DNA
  222. denaturing of DNA refers to
    how the helix separates into single strands
  223. supercoiling of DNA may be effected by antibiotics
  224. example of verticle transmission
    cell division
  225. the enzyme DNA primase is
    RNA polymerase
  226. Eukaryotic genomes are composed of non coding DNA while prokaryotes have mostly coding DNA
  227. semiconservative nature of DNA rep indicates that
    each daughter cell receives one parental strand and one newly synthesized strand
  228. most bacterial species DNA is
    - supercoiled
  229. 55 degree stage of PCR
    primers anneal to the denatured DNa strands
  230. organisms differ in genome size
  231. what shows the increasing number of genes
    gene, operon, regulon
  232. initiation of DNA rep in bacterial species
    is influenced by environmental factors
  233. X replication differs from rolling circle method of plasmid rep in that
    only X rep produces okazaki fragments
  234. a plasmid singly cut with a restriction enzymes that leaves cohesive ends
    can ligate to any DNA cut with the same restriction enzyme
  235. bacterial genome consists of
    the genome structure depends on species
  236. an example of DNA control sequence
    promoter region of a gene
  237. the plasmid encoded ParR and ParM proteins are involved in
    plasmid partitioning
  238. enzymes that regulate DNA supercoiling are called
    topo
  239. 95 degree strand in PCR
    dna strands denature
  240. E.Coli DNA pol III has what activity
    3-5' exonuclease
  241. this gene usually produces an RNA molecule that encodes a protein
    structural gene
  242. this regulates the expression of a structural gene
    DNA control sequences
  243. binding sites for regulatory proteins can do these 2
    activate or  inactivate that promoter
  244. prokaryotic genomes are made up of
    plasmids are made of
    • chromosomes
    • DNA
  245. functional units of DNA sequences include these 2
    • structural gene
    • regulatory sequences
  246. prokaryotic and plasmids can be circular or linear
  247. prokaryotes have large amounts of coding DNA while eukaryotes have small amounts of coding DNa and large amounts of non coding DNA
  248. noncoding regions of DNA in eukaryotes is composed of these 2 and their functions
    • enhancer sequences, needed to drive transcription of eukary promoters
    • DNA expanses that separate enhancers
  249. can function at large distances from the gene they regulate
    enhancers
  250. is the DNA seq immediately in front of a gene that is needed to activate the genes expression
    promoter
  251. how are all the genes in an operon situated and controlled
    • head to tail on the X
    • by a single reg sequence located in the front of the first gene
  252. the single RNA molecule produced from the operon contains all the
    info from all the genes in the operon
  253. a collection of genes and operons at multiple positions on the X can hold membership in a
    regulon
  254. horizontal gene transfer mechanism requiring cell to cell contact can transfer large segments of bac X
    conjugation
  255. transfer of X is non specific and requires recomb and sometimes it might take different amounts of time for it to occur
  256. adenine and thymine
    cytosine and guanine
    # of hydrogen bonds
    • 2
    • 3
  257. DNA with higher amounts of CG require higher denaturing temperatures due to it having 3 hydro bonds
  258. denaturing is much faster than renaturing since renaturing is random with a hit or miss type ordeal
  259. this is necessary step in decoding of genes to make proteins
    hybridization
  260. what causes the X of E.coli to have a - charge of the cyto
    all the phosphates in the backbone are unprotected and negatively charged
  261. bacteria nucleoid is distributed throughout the cyto instead if being compacted like the eukary
  262. boundaries of supercoiled loops are defined by anchoring proteins called
    histones
  263. supercoiling of one domain of DNA is maintained independently of other loops
  264. torsional stress of overwinding is relived how
    when DNA twists upon itself causing + supercoiling
  265. how are - supercoils formed
    if one end of DNA molecule is turned in the same direction as the helix, underwinding DNA
  266. - supercoiling is easier to separate because DNA is underwound
  267. + supercoiling DNA is harder to denature because
    it takes excess E to separate overwound DNA
  268. description of how spatial features of an object are connected to each other
    topology
  269. supercoiling changes topology of DNA
  270. enzymes that change DNA supercoiling are
    topoisomerases
  271. to maintain proper DNA supercoiling levels what must a cell do
    balance the activities of 2 types of topoisomerases
  272. this type of topo are usually single proteins
    have multiple subunits
    • 1
    • 2
  273. smallest genome known for free living microbes encodes
    480 proteins possible
  274. hydrogen bonding and interactions between the stacked bases hold together complimentary strands of DNA
  275. bacteria, eukary and most archae have - supercoiling
  276. these topo cleave 1 strand of a DNA molecule to relieve supercoil
    type 1
  277. this topo cleaves 2 strand of DNA and use ATP to introduce supercoils
    type 2
  278. circular microbial X replicates
    bidrectionally
  279. how is initiation of replication determined
    • DNA methylation
    • binding of specific initiator protein to the origin seq
  280. 3 things that can cause replication to begin again
    • origin is fully methylated
    • seqA dissociates
    • DNAA-ATP conc rises
  281. main replication pol known also as a molecular macine
    polIII
  282. function of exonuclease activity of polIII
    • cleaves the phosphodiester bond releasing improperly paired base from a growing chain
    • (once mispaired is fixed, polIII continues)
  283. polI enzyme enters after the rnase and syn the DNa patch using he 3OH end of the preexisting DNA fragment as a priming site
  284. template DNA is threaded through the replisome, the helicase pulls apart the 2 strands of the DNA helix, which causes
    DNA ahead of the fork to twist introducing + supercoiling

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