genetics TEST III (1).txt

how DNA is copied & maintained from cell division to the next.

• -Complimentary nature of the 2 strands of the double helix
• -suggested each strand of helix could serve as template to direct the synthesis of a new complimentary strand

• SEMI-CONSERVATIVE MODEL OF DNA REPLICATION
• EACH PARENTAL STRAND OF THE DOUBLE HELIX ACTS AS A TEMPLATE TO DIRECT THE ASSEMBLY OF A NEW DAUGHTER STRAND THAT IS COMPLEMENTARY TO ITSELF
• RESULT: EACH NEW MOLECULE OF DNA WOULD CONSIST OF A PARENTAL & A DAUGHTER STRAND
• THUS: THE PARENTAL STRAND IS SEMI-CONSERVED IN THE REPLICATED STRUCTURE
• 

• PARENTAL STRANDS ARE FULLY CONSERVED IN THAT THE ENTIRE DNA MOLECULE SERVES AS A TEMPLATE FOR A NEW MOLECULE COMPRISED OF ONLY NEWLY SYNTHESIZED DNA
• 

• LARGE AMOUNTS OF DNA
• THE ORIGINAL DOUBLE HELIX GETS BROKEN DOWN INTO MANAGEABLE SIZED FRAGMENTS
• EACH OF THESE FRAGMENTS, THEN SERVES AS TEMPLATE TO MAKE NEW DNA
• REASSEMBLE THE FRAGMENTS AFTER REPLICATION IS COMLETED
• LEAST LIKELY MODEL B/C THERE CAN BE ERRORS IN ASSMEBLY PROCESS
• 

• -5 YEARS AFTER STRUCTURE OF DNA
• CARRIED OUT A SET OF EXPERIMENTS IN WHICH THEY COULD LOOK FOR CORRECT MODEL OF DNA REPLICATION
• NEEDED A WAY TO DISTINGUISH B/W PARENT & DAUGHTER DNA
• USED HEAVY 15N & LIGHT 14N ISOTOPES OF NITROGEN TO LABEL THE NITROGENOUS BASES OF DNA
• 

SEMI-CONSERVATIVE

• DIFFER LARGELY BASED ON TEMPLATE
• *TYPE OF TEMPLATE
• *# OF REPLICATION ORIGINS
• 1.THETA REPLICATION
• 2. ROLLING CIRCLE REPLICATION
• 3. EUKARYOTIC DNA REPLICATION

INDIVIDUAL UNIT OF REPLICATION

• POINT ON CHROMOSOME WHERE REPLICATION ORIGINATES
• *EACH REPLICON CONTAINS A SINGLE ORIGIN OF REPLICATION

• REGION OF UNWINDING OF DOUBLE HELIX
• *EXPOSES ssDNA THAT CAN BE COPIED
• *CENTERED OVER THE ORIGIN

SITE OF ACTIVE REPLICATION AS IT MOVES DOWN REPLICATION TEMPLATE DURING COPYING STEPS

• NAMED AFTER THE GREEK LETTER
• COMMON TO CIRCULAR GENOMES
• EXAMPLE: BACTERIAL CHROMOSOME
• GENERATES A REPLICATION INTERMEDIATE THAT RESEMBLES THETA
• 1ST DESCRIBED BY JOHN CAIRNS
• EXP:
• *GREW E.COLI IN PRESENCE OF TRITILATED THYMIDINE (RADIOACTIVE)
• *SAMPLED THE CELLS AT VARIOUS TIMES OF REPLICATION PROCESS
• EACH SAMPLE WAS "LYSED" OPEN ONTO A GLASS SLIDE - RELEASE THE DNA
• *EXPOSED SLIDE TO X-RAY FILM OR PHOTOGRAPHIC EMMALISM - THE "HOT" DNA WILL BE CAPTURED IN A PHOTOGRAPHIC IMAGE
• *LOOK AT IMAGE OF DNA

• CONCLUSION:
• E.COLI CHROMOSOME HAS SINGLE ORIGIN OF REPLICATION=SINGLE REPLICON
• PROPOSED:
• DNA UNWINDS @ THE ORIGIN OriC (CHROMOSOMAL) - ALWAYS MEANS REPLICATION
• --GENERATES LOCALIZED REGION OF ssDNA THAT CAN NOW SERVE AS TEMPLATES TO DIRECT THE SYNTHESIS OF A NEW, COMPLEMENTARY DAUGHTER STRAND

• -PRESCOTT SHOWED E.COLI REPLICATION IS BIDIRECTIONAL
• 

• -COMMON TO CIRCULAR GENOMES
• -UNIDIRECTIONAL REPLICATION
• EX:
• *CONJUGATION & REPLICATION OF F FACTOR
• * BACTERIOPHAGE THETA REPLICATION
• 

• Linear DNA template
• A lot larger genome
• Utilizes multiple origins of replication

• **the human genome has
• 20k-30k origins
• If there was use of only a single origin

• Take 5-7 days
• Reality 3-4 hours

• Proceeds bidirectional
• Replication rate ~500-5k
• bp/min

• E.coli replicates
• ~1000bp/second

A lot slower in eukaryotes

• In large part due to all of the DNA packaging into nucleosomes
• 1st have to disassemble the nucleosomes to expose the ssDNA template & then reassemble after copying

Components of DNA replication

• ssDNA template
• dNTPs
• Primer
• MgH
• DNA polymerase

• -ssDNA TEMPLATE
• -dNTPs
• -PRIMER
• -MGH
• -DNA POLYMERASE

• A short chain of nucleotides
• that are complementary to the template DNA (base pairs w/the ssDNA template)
• *purpose of the primer is to
• provide a 3'OH group to which DNA polymerase can add a base to via a phosphodiester bond
• ALL DNA polymerases require
• a pre-existing 3'OH group

• MgH ->metal co-factor that stabilizes the DNA
• DNA polymerase ->enzyme that carries out DNA synthesis

• SIMPLY THE JOINING OF NUCLEOTIDES IN A POLYNUCLEOTIDE CHAIN BY LINKING THE NUCLEOTIDES W/A PHOSPHODIESTER BOND
• *THE PRODUCT IS COMPLEMENTARY & ANTI-PARALLEL TO THE TEMPLATE STRAND
• *DIRECTION OF SYNTHESIS IS ALWAYS 5'-3'
• 

• DNA replication in E.coli
• begins @ the Ori-C
• Ori-C
• -245bp region of the chromosome

• Contains 2 sets of tandem
• repeats

• -INITIATION
• -UNWINDING
• - PRIMING
• -ELONGATION
• -FINISHING TOUCHES

the series of 9-mer repeats

• Results in coiling of the DNA around these proteins (requires ATP for energy)
• This coiling puts a torsional strain of the double helix
• This tension needs to be released

• Unwinding ->to release the tension caused by coiling w/HU
• & Initiator protein

• Get unwinding @ the weak A/T
• pairing of 13-mers repeats
• Generates your ssDNA
• template
• 
• This unwound state is stabilized by single stranded DNA binding proteins (ssb proteins)

• Keeps the replication origin
• open for DNA synthesis
• Prevents the helix from
• reforming
• Further unwinding of the double helix by the enzyme-->

• Helicase->enzymatically breaks the H-bonds of the double helix
• as replication proceeds

*helicase binds to ssDNA @ the replication fork

• -breaks the H-bonds
• 

• -removes
• these supercoiled knots that result from helicase unwinding

Removes 2 supercoils @ a time

• As the DNA unwinds
• -supercoiled knots are formed in front of the replication fork

• carried out by the enzyme primase (a special
• type of RNA polymerase)

• Primase binds to the helicase @ the replication fork & sets down a short oligonucleotide primer of RNA
• bases that are complementary to the template DNA
• Promer provides the 3'OH
• group for DNAP to add DNA bases to
• **all DNA synthesis begins
• w/a short stretch of RNA (to be removed later & replaced w/DNA)

• ****ALL DNAPS REQUIRE A PRE-EXISTING 3'OH GROUP BUT RNAPS DON’T
• HAVE THIS REQUIREMENT

template

• **once DNA synthesis is
• primed, replication proceeds in a 5'-3' direction until a termination signal is encountered or 2
• replication forks meet
• ***elongation is carried out
• by DNAPs

• Synthesize new strands of DNA that are
• complementary & anti-parallel to the template
• By
• definition they have 5'-3' polymerase activity
• Use dNTPs
• as substrates for synthesis
• Require a
• primer to initiate synthesis

(unlike the RNAP primase)

• Catalyze the formation of a phosphodiester
• linkage b/w the 3'OH of 1 nucleotide & the 5' PO4 of the incoming, joining nucleotides

• -DNAPI, DNAPII, DNAPIII
• -IN ADDITION TO THE (1) 5'-3' POLYMERASE ACTIVITY, SOME DNAPs MAY HAVE ADDITIONAL ACTIVITIES (2) 3'-5' EXONUCLEASE ACTIVITY (INVOLVED IN PROOFREADING/CORRECTING MISTAKES IN SYNTHESIS) (3)5'-3' EXONUCLEASE ACTIVITY (INVOLVED IN REMOVING THE RNA PRIMERS & REPLACING W/DNA

ENZYME THAT BINDS TO THE ENDS OF DNA MOLECULES & REMOVES/DEGRADES NUCLEOTIDES (NUCLEASE)

• BINDS TO THE 3' END & REMOVES NUCLEOTIDES BY MOVING IN A 3'-5' DIRECTION = PROOFREADING ACTIVITY
• -BACKTRACKS & REMOVES THE BASES 3'-5' INCLUDING THE MISINCORPORATION
• 

• -PRIMARY FXN
• *MAIN WORKHORSE OF DNA REPLICATION.
• *ACTIVITY @ THE REPLICATION FORK ->SYNTHESIZED THE DNA
• -5'-3' POLYMERASE - YES
• -3'-5' EXONUCLEASE - YES (PROOFREADING)
• - 5'-3' EXONUCLEASE - NO

• -PRIMARY FXN
• *REMOVES THE RNA PRIMERS (W/5'-3'EXONUCLEASE) & REPLACES W/DNA (5'-3'POLYMERASE)
• -5'-3' POLYMERASE - YES
• -3'-5' EXONUCLEASE - YES
• -5'-3' EXONUCLEASE - YES

• -PRIMARY FXN
• *POST-REPLICATION REPAIRS/PROOFREADING ->CATCHES THOSE MISTAKES NOT CORRECTED BY DNAP III
• -5'-3' POLYMERASE - YES
• -3'-5' EXONUCLEASE - YES
• -5'-3' EXONUCLEASE - NO

• The strands of the double helix areanti-parallelBut, DNAsynthesis occurs only 5'3'Therefore,DNA synthesis proceeds in opposite directions (physically not biologically5'-3') on the parental strandsOne strandis said to be synthesized - continuously = leading strandThe otherstrand is synthesized - discontinuously = lagging strandThe lagging strand is madein a series of fragments = Okazaki fragment
• 

• Removal of the RNA primers
• & replacing them w/DNA =DNAP I

• Chews away the RNA 5'-3'
• while replacing w/DNA 5'-3'

• Uses the 3'OH group of the
• proceeding Okazaki fragment to initiate synthesis

• Joining of the Okazaki fragments on
• the lagging strand

• DNA ligase=molecular glue
• ->joins Okazaki fragments

• Joins 2 DNA fragments w/a
• phosphodiester linkage
• *but it cannot add bases
• (not a polymerase)

DNA ligase seals the nick

• ACCURACY
• ~OVERALL ERROR RATE OF 1MISTAKE/BILLION BASES
• ~DNAPIII MAKES MISTAKES ~1/1000000BASES
• ~PROOFREADING ACTIVITY CORRECTS SOME OF THESE MISTAKES = 1/1000000 CHANCES
• ~POST-REPLICATIVE REPAIR (DNAPII) - 1/BILLION MISTAKES

• (ASSOCIATED W/CELLULAR AGING)
• -THE CHROMOSOME GETS SHORTER W/EACH REPLICATION/DIVISION CYCLE

• THE BODIES WAY OF FIGHTING BACK AGAINST CHROMOSOME SHRINKAGE
• -AN ENZYME THAT BINDS TO THE ENDS OF LINEAR CHROMOSOMES (TELOMERES) & ADDS ON TANDEM COPIES OF THE TELOMERIC REPEAT SEQUENCE
• ~IT FIGHTS BACK AGAINST SHRINKAGE BY ELONGATING & ADDING MORE REPEATS
• -RIBONUCLEIC PROTEIN (RNA COMPONENT & PROTEIN COMPONENT)
• ~SPECIFICALLY RNA DEPENDENT DNAP
• ~USES AN RNA TEMPLATE TO DIRECT THE SYNTHESIS OF COMPLEMENTARY DNA STRAND
• ~IT USES ITS OWN RNA AS THE TEMPLATE
• 

• ~HOMOLOGOUS
• ~NONHOMOLOGOUS

• COMMON IN GERMLINE CELLS THAT ARE UNDERGOING MEISOSIS (MEIOSIS I)
• ~THE MECHANISM IS NOT COMPLETELY UNDERSTOOD-BUT THERE IS A MODEL FOR THIS RECOMBINATION BASED ON OBSERVABLE INTERMEDIATES - HOLLIDAY MODEL

NO HOMOLOGY INVOLVED IN THIS GENETIC EXCHANGE -> COMMON IN SOMATIC CELLS THAT DO NOT GO THROUGH MEIOSIS

• MODEL OF HOMOLOGOUS DNA RECOMBINATION BASED ON OBSERVABLE INTERMEDIATES
• 1.RECOGNITION & ALIGNMENT
• 2.SINGLE-STRAND BREAKAGE
• 3.CROSSING OVER & STRAND INVASION
• 4.JOINING OF THE END (DNA LIGASE)
• 5.BRANCH MIGRATION & HETERODUPLEX FORMATION
• 6.CLEAVAGE & RESOLUTION

• 
• -HAVE VERTICAL AND HORIZONTAL CLEAVAGE PRODUCTS

• to store the cellular info in a stable form ->such that it isn't
• lost when a cell divides & replicates
• The DNA sequence is a code that when deciphered directs
• the assembly of amino acids intoproteins
• It is the proteins that carry out all the
• cellular/biochemical processes.

• -->
• proteins control the phenotype

• Although DNA stores all this
• info -->it is NOT the primary template used for protein synthesis.

• -the term used to describe the process of taking the info in DNA, & using it to direct the synthesis of proteins.
• -there are 2 main processes in gene expression
• *TRANSCRIPTION
• *TRANSLATION

the synthesis of ssRNA (mRNA) from a DNA template -> produces an mRNA molecule that is complementary & anti-parallel to the DNA strand

• 5'-3' synthesis of ssRNA from a dsDNA template
• *Although the mRNA is transcribed from dsDNA template,

--->only 1 strand of the double helix actually serves as the template

• mRNA has the same polarity as the non-template strand
• mRNA has the same sequence as
• the non-template strand except T are replaced by U

• b/c of this = the nontemplate strand is also referred
• to as the "sense" strand

Template strand = antisense

Process of transcription is similar to replication

------>5' - 3' synthesis of a polynucleotide chain from dsDNA

• synthesis of a polypeptide (protein) from an
• mRNA template

• --->FLOW OF GENETIC INFORMATION
• 

• Messenger RNA
• (mRNA) - encodes the amino acid sequence that
• determines a protein

• mRNA is copy of the gene coding region
• Define protein sequence

• Transfer RNA
• (tRNA) - a functional RNA molecule that serves as an
• adapter that links the amino acids with the correct coding in Mrna
• Ribosomal RNA (rRNA) - structural & functional compiment of ribosomes

Small nulcear RNAs (smRNA) - involved in splicing eukaryotic genes

• Elongation
• Termination

• RNA coding sequence - gene coding region
• Terminator -where transcription terminates

• 
• 

-gene coding region

• begins with the
• RNAP binding to the promotor

• The core enzyme of the RNAP is a tetrameric protein
• comprised of 4 polypeptide subunits

• 2 molecules of alpha subunit
• 1 molecule of beta subunit
• 1 moluecule of beta prime
• subunit
• Shorthand Α2ββ'

• Core RNAP - responsible for catalyzing elongation by
• joining rNTPs with a
• phosphodister bond

Enzymatic component of the RNAP

• Inititation
• is a 2-step process

Loose binding of the holoenzyme to the -35 site

This binding facilitates localized unwinding at the A/T rich pribnow box (-10 site)

Creates a "bubble" of ssDNA template

• Results in tight binding to the now available ssDNA
• Centered over the -10 site

σ- factor associates w/the core RNAP to form the HOLOENZYME (σ2ββ'α)

• THE
• SPECIFICITY OF BINDING IS DETERMINED BY THE SEQUENCE OF DNA W/I THE PROMOTOR

• 
• *IF YOU MUTATE OR
• CHANGE THESE CONSERVED SEQUENCES -> REDUCE OR ABOLISH TRANSCRIPTION

• *NOTE: PRIBNOW BOX =
• A/T RICH

• WEAK
• h-BONDS

• FACILIATATES
• UNWINDING OF HELIX TO EXPOSE ssDNA template for transcription

• Transcription proceeds in 5'-3' direction until a
• termination signal is met
• After 8-10 nt have been
• synthesized the sigma factor falls off, & the core RNAP (catalytic
• component) continues on w/transcription until the end

• ***the
• sigma factor is recycled to form another holoenzyme that can begin a second
• round of transcription from the same template

• ***the association of the mRNA w/the DNA template is temporary (unlike replication)
• As the RNA synthesized, it dissociates from the template -> DNA helix reforms behind it
• -> RESULTS: the promotor becomes available again for a sequential round of transcription

-> Multiple mRNAs can be transcribed simultaneously from the same template

• (drawing in book) - looks like a christmas tree
• -> each of these
• mRNAs can then be simultaneously translated into proteins by the ribosomes

• Can
• have multiple ribosomes along same mRNA

• Very
• efficient system for making lots of protein

• ->Polyribosome/polysome
• => simultaneous translation of a single mRNA by multiple ribosomes

• Rate of
• transcription is approximately 20X slower than replication

• Transcription
• is 50nt/sec

• Replication
• is 1000nt/sec

• Slower
• rate due in part due to single stranded mRNA forming secondary structures due
• to complementary base pairing - slows down to forward synthesis

• mRNAs have a short half-life and they get degraded
• quickly

• So even if you make a mistake it's not going to be
• around that long for a bad protein to be made
• And then right behind that
• bad mRNA is another copy being synthesized which would probably be a good
• copy

• *The lack of RNA
• proofreading is an advantage to RNA viruses b/c it allows them to evolve
• quickly

• The termination signal is found downstream of the Gene
• Coding Region

• In bacteria there are 2 mechanisms of transcription
• termination:
• Self-termination/Rho-independent
• Rho-dependent

• Based solely on the characteristics of the sequenceat the termination site
• A non-enzymatic dissociation of mRNA from the DNA template
• Template characteristicsG/C-rich region that is followed by a poly-A tail (6or more Adenine in a row)
• G/C -rich region ispalindromic (inverted repeat)-facilitatescomplementary pairing/hairpin formation in the transcribed mRNA.

• Enzymatic dissociation of mRNA from the DNA template
• Rho- enzyme= HELICASE

• DISSOCIATES THE mRNA from the template by
• enzymatically breaking H- bonds

• G/C rich region - but not followed by a poly A tail
• Upstream of this region is a
• rut-site (rho-utilization)

• Site where rho binds to the mRNA
• Once bound, rho moves down
• the transcript in a 5'-3' direction following the RNAP

• When the RNAP reaches the G/C rich region ->slows
• down (harder to transcribe through
• the triple bonds of G/C pairs)
• This allows rho to catch-up
• & it breaks the H-bonds holding the transcript template - termination

• More proteins involved = these proteins are called
• transcription factors (tfs)

• A transcription factor is any protein that influences
• the rate of transcription in either an increase or decrease in the amt of mRNA made

More RNAPs

RNAP II that transcribes mRNA

• The mRNA must go through several processing steps
• (post-transcriptionally) beofre it is ready for translation pre-mRNA----->mature mRNA
• Transcription takes place in
• the nucleus, while translation occurs separately in the cytoplasm =UNCOUPLED

• For prokaryotes there is no nucleus and transcription
• and translation occur simultaneously or
• COUPLED

Euk. Transcription units =monocistronic

• Prok.
• Transcription units = may be polycistronic

expressed DNA

interventing DNA

• elements that when bound by transcription factors called ACTIVATORS serve
• to increase the rate of
• transcription
• They don't have to be partof the promoter In fact they can be located upstream, downstream oreven w/I the gene itselfAnd they can act over greatdistance

positive transcription factor - they act bystabilizing RNAP binding to the promotor

elements similar to enhancers w/regards to position

• Except when bound by tfs called REPRESSORS they
• decrease or inhibit transcription by the RNAP

Destabilize RNAP binding to the promoter

• -5' CAPPING
• -3'POLYADENTATION
• -SPLICING

• Shortly after transcription
• begins, a 7-methyl guanosine cap is added to 5' end of mRNA (McG)
• Serves 2 functions

• Protects the 5' end from
• degradation
• Serves as the Ribosome
• Binding Site (RBS) for translation

• (*no cap->no
• translation->no protein made)

• the addition of 300-500 adenines to the 3' end
• of transcript

Serves 2 fxns

• Protects the 3'end from
• degredation
• Involved in transcriptional
• termination

Ends the transcript lenth

• The enzyme responsible =
• poly A polymerase=PAP

• A template INDEPENDENT
• polymerase

• **adds adenines w/out a
• template of thymines to direct it

• At the 3' end of pre-mRNA
• -->there is a conserved consensus sequence that is recognized by a complex of cleavage proteins

the removal of the non-coding intron & the joining of the coding exons

End result = mature fxnal mRNA that is ready for transport & translation

• Splicing is very important
• ->must be done accurately ->to the exact base level

• If you improperly join the
• exons->non-fxnal mRNA ->bad, non-fxnal protein
• Precise slicing is directed
• by specific DNA sequences in the mRNA that are located @ the exon/intron boundaries

• Introns may protect the important coding regions of genes from errors in homologous meitoic recombination
• Exon shuffling -rearrangement of coding exons @ the DNA level

Rearrange the exons to create new genes

Exons are typically functional domains of a protein

• Phosphorylation activity
• DNA binding domain
• Nuclease domain

Inherited changes - evolutionarily important

• Alternative
• splicing/deferential splicing

• The rearrangement of exons @ the RNA level->occurs during splicing of the mRNA
• Result: create more than one mature mRNA from a single pre-mRNA

Single transcript can actually end up coding for more than one protein, depending on how the exons are joined