MCB 150 Exam II Outline-Bacteria.docx

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Molecular and Cell Biology
MCB 150
Brad Mehrtens

Bacteria DNA Organization  Circular chromosomes: found in bacterial and archael cells o Cytoplasm of prokaryotes BACTERIA DNA PACKING:  Supercoil o Topoisomerase: cutwind/unwindreseal DNA  Induce/relax supercoil  Do not add or subtract base pairs  Reversible  Molecule twists and is glued exactly in the same sequence to take up less space  TYPE 1: o Cuts 1 strand  Twists one strand, wraps loose end around and twists around and touches the same base pairs  Save space, same sequence o Cuts 2 strands  Cuts two strands, twists the whole molecule and glues it back together (2x twist)  Eukaryotes have TOPOISOMERIASES  Not for supercoiling and packing DNA  They use them to relax the DNA strands before helicase can unzip it  Nucleus is NOT homogenous  It is super organized and must get 2 meters of DNA into 5-8 microns of diameter o must do more than supercoiling DNA REPLICATION Primase: (e.coli) o Physically attached to the other replication machinery o Puts in primers  3 H bonds form between ie. Guanine base and cytosine base (in the first part) (DNA polymerase cannot do this)  The 1 primer keeps its 3 phosphates (and does not break off 2) because there is no previous nucleotide so no energy needs to be released to bond it to a prior nucleotide  You cannot just start adding DNA after this even though you have a free 3’ OH group because the interaction between G and C is kind of flimsy (only hybridization)  The next bases put down by primase are hybridized AND do a condensation reaction (break off 3 phosphates and use the energy to attach it to the base before itstronger)  Gives DNA POLYMERASE III the 3’ OH group to link to once you allow the incoming base to hybridize  DNA pol III cannot just do the hybridization part  Hybridization is not covalent + is not stable DNA POLYMERASE III: primary DNA synthesizing enzyme with the highest processivity  Hybridizes and condense  Primary DNA synthesizing enzyme for e. coli o Most of the phosphodiester linkages in e. coli put down by DNA POL III  Processivity: puts in a lot of bases in a row very fast without falling off or being pulled off  Every okazaki fragment must be primed  1 okazaki goes until it runs into the primer of the leading strand nd st  2 okazakiprimer of the 1 O. Strand Third Problem: RNA bases in the genome  DNA POLYMERASE I: nibbles out RNA base pairs and leaves a space to add DNA bases o REMOVES AND REPLACES o Starts at 5’ o makes a nick and removes RNA bases o doesn’t move as fast, but can multi task  now you have fragments  shifts nick over and polymerase can start at the free OH at 3’  DNA POL I has multiple functions: o Creates phosphodiester linkages (in one active site)  DNA base addition (replacing primers) o Also breaks phosphodiester linkages  Exonulcease: removes nucleotide from the outside end of the chain  Moves from 5’ to 3’ end o 5’ to 3’ exonuclease: DNA POL I  one of its active sites  primer removal  “nick” from pol I still remains o must create a covalent bond between the 3’ and neighboring 5’ end o endergonic reaction (needs energy) o cannot use the energy from the last base on the 3’ end to link it to the next base on the 5’ end because when you break phosphates off, you use the energy to link it to the base BEFORE it, not after it. o Energy to drive endergonic rxns comes from ATP DNA LIGASE: enzyme will use a molecule of ATP to form that missing covalent bond  Not a polymerase and cannot fill in gaps  It can only seal NICKS by using energy from ATP hydrolysis Cannot have RNA in the genome because when you go through replication again, the DNA template cannot read them because its made of RNA sugars  Topoisomerases necessary to relax the DNA strands (relax supercoiling) before Helicase unzips so they are able to do so o DNA GIRASE DNA TRANSCRIPTION MOST genes are found in 1 or 2 copies of the cell & this is not much template for making so many proteins Amplification: a lot of protein must be made  If ribosome’s could directly read DNA, many ribosome’s would be just waiting for their turn to get there (NOT EFFICIENT)  Instead: one molecule reads the protein and make a bunch of secondary molecules MESSENGERS o Lots of ribosome’s work on the each of the messengers to AMPLIFY the signals  IE) 270 million copies of hemoglobin in every one RBC  4 polypeptide chain in every molecule ( a lot of A.A.)  if it were possible for ribosome’s to have direct access to DNA, it could not make 270 million copies in efficient amount of timesignal must be amplified Transcription: E.Coli RNA intermediate 1) Promoter Recognition 2) Initiation 3) Elongation (elongate initiated fragment) 4) Termination (stop when told when gene is done)  Production of an RNA molecule from the information in a DNA template o Each RNA will be read by a ribosome  DNA DEPENDENT RNA SYNTHESIZING ENZYMES: RNA POLYMERASES o Does the same thing as primase, but in different spots o E. coli  Has an RNA polymerase that has in total 6 different subunits  When all 6 subunits work together e.coli rna polymerase HOLOENZYME  Holoenzyme:  Core Enzyme: holoenzyme MINUS the sigma subunit o sigma can be attached/detatched Unlike DNA replication where it goes until it is DONE (everything copied), Transcription does NOT transcribe the entire genome into RNA  takes the part of the genome that it needs ONLY o start points and stop points o units of information: GENES o many genes in bacteria o not as many genes in humans  most DNA in humans are not templates o looks for specific regions of information (individual genes) o Start Signal: PROMOTER sequence  Sequence of bases in the DNA that gets recognized by RNA POLYMERASE  POL knows that work must be done and goes until the TERMINATOR region Promoter Recognition Bacteria: start signal, coding region (message itself) , stop signal Purple: promoter Gold: within a promoter regionextremely important “hot spots”  +1 base= the first base in the transcript o increase in positive way: DOWNSTREAM coding region  NEGATIVE numbers: UPSTREAM o -10 site: 10 bases away from where you start transcription o -35: 35 bases away from where you start transcription  -10 and -35 sites: CONSERVED region among e. coli  consistency between others  gold sections: very similar sequences  CONSENSUS: most occurring base in conserved region  if all had the same baseinvariant o The e. Coli RNA POLYMERASE is looking for -35 (TTGACA) and -10 (TATAAT) bases away or else it’s not recognized as a gene o The closer the bacterial promoter is to the -10 and -35 conesnsus sequence, the better RNA polymerase will bindMORE RNA  There can be some deviations, but it will be less well recognized  More likely hood that RNA polymerase will skip right over you  -10 SITE: TATAAT  -35 SITE: TTGACA  have to be careful to find one sequence at a random spot, so you must have the SECOND conserved region to back it up
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