genetics 4

two strands one old, one new

incorrect mode

one new strand, one old strand, coiled together

correct mode of replication

mixture of old and new on one strand

incorrect mode

-Meselson- Stahl experiment

-DNA replication is semi-conservative

-each new strand of DNA consist of one old strand and one newly synthesized strand

demonstrated that DNA replication is semiconservative in eukaryotes

begins at one origin of replication

bidirectional

length of DNA that is replicated following one initiation event at a single origin

catalyzes DNA synthesis and requires a DNA template and all four dNTPs

no

yes

yes

polymerase I

primer

none

proof reading, fixes wrongly matched polymers

removing the primer

the synthesis that fills gaps produced during synthesis

5'-3' polymerization essential in vivo

3'-5' exonuclease activity allows proof reading

10 subunits

not involved in repair

• Step one
• DnaA intial steps in unwinding
• DnaB and DnaC further opens and destabilizes the helix
• Energy from hydrolysis of ATP used to supply these proteins

Stabilize the open helix

Step one- unwinding the helix

proteins that open DNA structure

DnaA, DnaB, DnaC

• Step 2
• The unwinding creates super coiling
• DNA gyrase stops super coiling

• Stops supercoiling
• A DNA topoisomerases

• Step 3
• Elongation requires a primer with a 3'-hydroxyl group
• Primase used

• Enzyme
• Synthesizes an RNA primer that provides the free 3' hydroxyl required by DNA polymerase 3

• Step 4
• Replication fork moves, one strand is used for template for continuous DNA synthesis
• Opposite strand undergoes discontinues DNA synthesis

• Undergoes continues DNA synthesis
• Enlongated

• Undergoes discontinues DNA synthesis
• Synthesized into Okazaki fragments-  (small fragments each with an RNA primer

• DNA polymerase 1
• Removes the primers on the lagging strand
• Replaces the missing nucleotides (fills gaps)

• Step 6
• Fragments of lagging strand joined by DNA ligase

fills gaps, binds lagging strands of DNA

Fix any possible mistakes

• DNA polymerase 3
• Single-stranded binding proteins
• DNA gyrase
• DNA helicase
• RNA primers
• DNA ligase

prevents the core enzyme from falling off the template during DNA synthesis

• More complex
• More DNA than prokaryotic cells
• Chromosomes are linear
• DNA complexed with proteins
• Multiple origins of replication=faster

involved in efficient initiation

pol alpha and gamma

• Low processivity
• Synthesis of RNA primers during initation on leading and lagging strand

length of DNA synthesized by an enzyme before it dissociated from the template

• Found on ends of linear chromosomes
• Long stretches of short repeating sequences
• Preserve the integrity and stability of chromosomes
• Problematic to replication
• Can cause gap in replicated strands

• Directs synthesis of the telomere repeat sequence to fill the gap
• Ribonucleoprotein with RNA
• Serves as a template for the synthesis of its DNA complement

• Genetic exchange at equivalent position along two chromo. with substantial DNA sequence homology
• AKA general, homologous, recombination
• Two chromo. similar sequences

• Endonuclease nicking
• Strand displacement
• Ligatioin
• Branch migration
• Holliday structure

• Duplex seperation
• Miosis
• Holliday junction

sequences similar but mixed

Ab Bb

• Consequence of DNA recombination
• Nonreciprocal genetic exchange between two closely linked genes

RNA

protein

A U C G

nontemplate strand

template strand

• Specifies one amino acid
• Provide 64 codes to specify the 20 amino acids
• Contains start and stop signals

• UGA
• UAA
• UAG
• Do not code for any amino acid

AUG

• Insert 1 or 2 nucleotide, protein sequence completely changes
• Insert multiples of 3 it does not completely change

• Determines specific codon assignments
• Ribosomes bind to a specific codon

• Many amino acids specified by more than one codon
• Only tryptophan and methionine are encoded by a single codon

3 and 1 position in mRNA and tRNA can be modified

overlapping genes, 2 different gene sequences

DNA and proteins

transcription

at the same time

• Different times
• Different places

• Directs synthesis of RNA using DNA template
• No primer required in initiation
• Uses ribonucleotides instead of deoxyribonucleotides

• RNA polymerase enzyme holoenzyme
• Promoter sequence on DNA template
• Ribonucleotides

• RNA polymerase binds to promoter
• Start sequence TTGACA and TATTAT

sigma dissociates and elongation begins with core enzymes

• Transcription terminates due to hairpin formation in the RNA
• Some depend on Rho termination factor

• 1. RNA polymerase binds to promoter
• 2. short sequence of DNA is unwound
• 3. RNA synthesis begins
• 4. sigma factor dissociates
• 5. RNA elongates

• Eukaryotes
• Transcription occurs in nucleus and not coupled translation
• Requires chromatin remodeling
• mRNA processing to produce mature mRNAs
• RNA polymerase (I,II,III)
• Promoter needed
• Transcription factors
• Enhancers and silencer

• 1 promoter
• 2.RNA polymerase
• 3.transcription factors
• 4.enhancer and silencer
• 5. ribonucleotides

• TATAA box -35 from start site
• GGCCAATCT box -80from start site

• I, produces rRNA
• II, produces mRNA,snRNA
• III, produces 5S rRNA , tRNA

1. general transcription factors

2. Gene-specific transcription factors

required for all RNP II mediated transcription and help RNA polymerase II bind to the promoter and initiate basal level transcription

influence the efficiency or the rate of RNP II transcription

• Can be upstream, within or downstream (anywhere)
• Can modulate transcription from a distance

• RNA polymerase II + general transcription factors
• 35 subunit at promoter

• Associates and enables either positive or negative regulation of initiation
• 20 subunits after initiation complex

• Regions of initial RNA transcript that are not expressed in amino acid sequence
• Removed by splicing and exons joined by mature mRNA

spice out Pre-mRNA introns

• Multiple ways to put genes together
• Different exons are included in mRNA
• Increases genetic expression

• Carboxyl group
• Amino acid
• R (radical) group -refers to specific chemical properties

• Forms by dehydration reaction between the carboxyl group of the amino acid and the amino group of another
• Bonds amino acids

polymerization of amino acids into polypeptide chains

• Amino acid
• Messenger RNA (mRNA)
• Ribosomes
• Transfer RNA (tRNA)

70S

80S

• E-site: exit site
• P-site: peptidyl site
• A-site: amnioacyl site

CCA sequence at 3' end is binding site for amino acid

actives tRNAs with the appropriate amino acid

• Initiation
• Elongation
• Termination

• Small and large ribosomal subunits
• Initiation factors
• Charged initiator tRNA
• Mg
• GTP

• AUG
• Before it is AGGAGGU

AGGAGGUGAAUG

• Requires both ribosomal subunits with mRNA, form P (peptidyl) site and A (aminoacyl) site
• Charged tRNA enter A site
• Peptidyl transterase- catalyzes peptide bond between amino acid on tRNA A site and the peptide chain bound to tRNA P site
• Uncharged tRNA moves to E site
• tRNA bound to peptide chain moves to P site

• Stop codon
• UAG
• UAA
• UGA

take off polypeptide chain from tRNA and release it from translation complex

• Ribosomes are larger than in bacteria
• Transcription and translation are in different locations at different times
• Ribosomes are with endoplasmic reticulum instead of free floating
• Ribosomes must have initiator sequence in proper order (Kozak sequence)
• Requires more factors for initiation, elongation, and termination than bacteria

initiator tRNA in proper sequence

• Initation begins with methionine-not N-formyl-methionine
• tRNA:tRNAi met
• Initator tRNA bears unformylated methionine

Contain no Shine-Dalgarno sequence to show ribosomes where to start

requires more initation factors than eukaryotes

• Eukaryotes contain 2 release factors
• eRF1
• eRF3

mRNAs with several ribosomes translating at once

positive and negative conditions

• Upstream
• cis-acting

transacting elements

• An induced system
• Enzymes responsible for lactose metabolism are induced
• Lactose is the inducer

• Three structural genes lacZ, lacY, lacA
• Upstream regulatory region with a promoter and operator

• lacY
• enzyme that facilitates the entry of lactose into the bacterial cell

• lac A
• Involved in the removal of toxic by-products of lactose digestion from the cell 

• repressor molecule
• which is allosteric

• interacts reversibly with another mlc.
• Causes 3-D shape change and change in chemical activity

• =repressed
• Repressor binds to operator
• Blocks transcription
• No transcription
• No enzymes

• = induced
• No binding occurs
• Transcription proceeds
• Transcription > mRNA> translation > enzymes
• Operator-binding region altered when bound to lactose

• Active=always on
• nucleotide sequence of operator DNA is altered and will not bind with normal repressor

• Active=always on
• repressor protein is altered/absent and can not bind to operator region

• Repressed= not on
• Lactose binding region is altered, repressor always bound to operator

• Genetic info from one bacterium is transferred to another
• F+ cells are donor
• F- are recipients
• Fertility factor (F factor)

• F factor
• Can donate DNA during conjugation

• Represses expression of lac operon when glucose is present
• Used when there is enough lactose and glucose present

• repression of the expression of lac operon
• Happens when there is enough lactose and glucose

• Absence of glucose
• Presence of lactose
• CAP-binding site and RNA polymerase bind to promoter
• Max expression=respressor not bound to operator and CAP bind CAP-binding site
• Regulated by activator CAP

• CAP binding
• levels increase as glucose decreases

• repressed by glucose
• Catalyzes the production of cAMP
• Prevents CAP from binding when glucose is present