U3: C15: Molecular genetics Flashcards
DNA is deoxynucleicacid
ds, antiparallel double helix that exhibits base pairing bw complementary nitrogenous bases.
purpose is to store the genome
RNA is ribonucleic acid
- difference bw DNA
- Types or RNA
-It substitutes ribose for deoxyribose and uracil for thymine
it is usuually single stranded (ss) and exists in the forms of mRNA, tRNA, rRNA, and hmRNA
Helicase
Replication of the genome requires enzymes to seperate the strands (helicase)
and insert the complementary bases (DNA polymerase) in a 5’->3’ manner.
hnRNA -> mRNA in euk
Generation of eukaryotic mRNA from an hnRNA requires the addition of a 5’-guanosyl cap and 3’-poly-A tail. Introns are spliced out.
(exons exist in the RNA)
The genetic code is degenerate.
Multiple codons may code for the same amino acid.
This is one of the mechanisms by which silence mutations occur.
(usually first 2 nitrogenous bases dictate the amino acid) (ex: UCU & UCG both Serine Amino Acid)
Start and Stop Codons
Codon: mRNA (5’->3’)
Start Codon: AUG (Methionine Amino Acid)
Stop Codon: UAA, UAG, UGA (U -AG/AA/GA)
Translation Consists of 3 phases
- Initiation: AUG start codon tRNA anticodon TAC w/ amino acid Methionine.
- Elongation: tRNA anticodon addition to
mRNA (5’->3’)
- Termination: UAG/UAA/UGA stop codon
Post translational modifications may include
addition of covalent moieties : methylation, carboxylation, glycosylation
cleavage of large peptides before they are active
Viral Genomes are more diverse than the genomes of cellular organisms.
They may be single or double stranded, they may also be made up of DNA or RNA
Bacteria increase genetic diversity by..
- transduction
- conjucation
- transformation
Bacterial control at transcriptional level.
inducible and repressible systesms to control gene gene expression.
DNA composition
- Deoxyribose sugar (no hydroxy in C2)
- Phosphate group (phosphodiester bond @ C5 and C3)
- Nitrogenous base (@ C1)
ACGT = 2 1 2 1
2 Rings = Purines (Adenine and Guanine)
1 Ring = Pyrimidine (Cytosine and Thymine)
A = T (2 H bonds) Weaker
C = G (3 H bonds) Stronger
Transcription, Translation direction
5’ -> 3’
NTP’s can only be added to the 3’ end bcs of the hydroxy group.
Replication is the same way.
Semiconservative Replication
How do you tell the difference between the old strand and new strand?
New strand will not have methyl groups, the older strands will have methylation. Methylation is used to control gene expression.
Euk Replication Steps
- Mutliple origin of replications present
- each origin of replication has new DNA generation in both directions, creating replication forks.
- Enzymes
a. helicas: unwind helix, generating single stranded regions of DNA
b. single strand binding proteins (SSB): prevent the single stranded DNA from re H bonding with the other single strand (recall DNA is ds DNA)
c. DNA gyrase (a topoisomerase): relieves (torsion strain) overwound DNA by introducing negative supercoilds.
d. Primase: RNA polymerase, generates the RNA primer, which is what DNA polymerase recognizes.
e. DNA polymerase III: add dNTPs (nucleotides) to the growing strand starting from where the primer leaves off, synthesizing from 5’->3’ direction. Also proofreads new strand 5’->3’ direction.
f. DNA polymerase II: replace RNA primer with DNA, and proofreads new strand in 3’-> 5’ direction.
g. DNA ligase: Joins all the DNA together
http://cmapspublic2.ihmc.us/rid=1L3QS6XDK-2Q283Z-1PSM/DNA%20Replicaion.jpg
DNA vs RNA
- strand
- sugar
- base pairing
- location
- double stranded/ single stranded
- deoxyribose/ ribose
- AT, CG/ AU, CG
- Nucleus/ Nucleus and Cytoplasm
mRNA
(EUK vs PROK)
messenger RNA: takes genetic message from nucleus to cytoplasm sot that it can be translated into protein.
EUK: monocistronic: each mRNA molecule translates into only 1 product.
PROK: polycistronic: different proteins formed by starting translation at different positions on the mRNA.
Topoisomerases malfunction will lead to..
prevent DNA replication, by preventing DNA unwinding.
tRNA
transfer RNA. In cytomplasm, carries 20 amino acids, that are selected by different codons on the mRNA. tRNA has the anticodon.
rRNA
ribosomal RNA, synthesized in the nucleolus. Forms an integral part of the ribosomes that are used for protein assemply in the cytoplasm.
hnRNA
heterogeneous nuclear RNA (pre-mRNA), larger and includes riboproteins that mRNA does not have. It is the precursor to mRNA.
DNA: 5’-introns-exons-introns-exons-3’
pre mRNA:
cap-5’-introns-exons-introns-exons-3’-poly A tail
mRNA: cap-5’-exons-exons-3’-poly A tail
(DNA, premRNA, mRNA in nucleus)
(only mRNA leaves the nucleus)
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What is necessary for a piece of RNA to become a messagge?
- a 5’ guanosyl cap must be added
- a poly A tail must be added
- introns must be spliced out (exons exist)
Failure of all 3 steps lead to degradation of pre-mRNA
Heterochromatin
Heterochromatin is a tightly packed form of DNA, which comes in different varieties. These varieties lie on a continuum between the two extremes of constitutive and facultative heterochromatin. Both play a role in the expression of genes, where constitutive heterochromatin can affect the genes near them (position-effect variegation) and where facultative heterochromatin is the result of genes that are silenced through a mechanism such as histone deacetylation or siRNA through RNAi. Constitutive heterochromatin is usually repetitive and forms structural functions such as centromeres or telomeres, in addition to acting as an attractor for other gene-expression or repression signals. Facultative heterochromatin is not repetitive and although it shares the compact structure of constitutive heterochromatin, facultative heterochromatin can, under specific developmental or environmental signaling cues, lose its condensed structure and become transcriptionally active.[1] Heterochromatin has been associated with the di and tri-methylation of H3K9 in certain portions of the genome.[2]
Euchromatin
Euchromatin is a lightly packed form of chromatin (DNA, RNA and protein) that is rich in geneconcentration, and is often (but not always) under active transcription. Euchromatin comprises the most active portion of the genome within the cell nucleus. 92% of the human genome is euchromatic
Chromatin
Chromatin is the combination or complex of DNA and proteins that make up the contents of thenucleus of a cell. The primary functions of chromatin are 1) to package DNA into a smaller volume to fit in the cell, 2) to strengthen the DNA to allow mitosis, 3) to prevent DNA damage, and 4) to control gene expression and DNA replication. The primary protein components of chromatin are histones that compact the DNA. Chromatin is only found in eukaryotic cells: prokaryotic cells have a very different organization of their DNA, which is referred to as a genophore (a chromosome without chromatin).
mRNA
molecule in cells that forms part of the protein-synthesizing organelle known as a ribosome and that is exported to the cytoplasm to help translate the information in messenger RNA (mRNA) into protein.
(ribosome organelle and ribosome proteins are not the same thing)
nucleolus
where rRNA is synthesized and the assembly of ribosome subunits from rRNA and ribosomal proteins. Ribosomal proteins are synthesized in the cytoplasm and transported to the nucleus for subassembly in the nucleolus. The subunits are then returned to the cytoplasm for final assembly.
Translation
Prok vs Euk Gene regulation
- promoter
- sites for binding transcription factors
Prok
- several protein coding genes are transcribed coordinately under the control of a single promoter (operon)
- operators, are right next to the promoter (upstream): the regulatory system is very compact.
Euk
- Each protein coding gene has its own promoter, there are no operons
- enhancer or silencer far away from promoter by severl hundred base pairs, but due to 3D shape and folding, the enhancer and silencer are not that far from the promoter.
- transcription factor bound to enhancer, increases transcription
- transcription factor repressor binds to silencer, decrease transcription
enhancer/silencer can be upstream, downstream
Gene Expression Regulation at (include differences bw prok and euk)
- transcription level
- some genes are actively transcribed, while others are not. Activators and inhibitors modulate the transcprition of a gene.
- prok: promoter next to the operator = operon
- euk: no operon, promoter (TATA box) and enhancers (binding sites for transcription factors) far from promoter.
http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/negposreg.gif
Gene Expression Regulation at (include differences bw prok and euk)
- translation level
- some mRNA get translated more
1. prok: mRNA with better Shine-Dalgarno sequence.
2. euk: regulation by mRNA modifications- by adding more polyAs (longer mRNA life),
- modulating the translation machinary (phosphorylation of initation factors)
- storing mRNAs to be translted at a lter time (mRNA masking)
Negative vs positive gene regulation
http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/negposreg.gif
Prok DNA promoter Image
Euk DNA promoter Image
Location of transcription and translation control
PROK Transcription
(parts and enzymes)
Promoter: binding site for RNA polymerase II, site of transcrption inihitiation
Operator: binding site for regulatory proteins, usually a repressor.
http://cmapspublic2.ihmc.us/rid=1L3QS6XDK-2Q283Z-1PSM/DNA%20Replicaion.jpg
EUK Transcription
(parts and enzymes)
What does Translation require?
mRNA, tRNA, amino acids, ribsomes, and energy
mRNA (codon): 5’- AUG-> 3’
tRNA (anticodon): 3’- UAC -5’
(mRNA and tRNA are antiparallel)
Ribosome binding sites for tRNA
- A site: holds the aminoacyl-tRNA complex
- P site: binds to the tRNA attached to the growintg polypeptide chain.
Steps of translation
Each tRNA has a helper tRNA synthetase (enzymes that use GTP to bind amino acid to the tRNA)
- Initiation: In the presence of initiation factors, small ribosomes slides along the mRNA until it reaches a start codon (5-AUG-3). aminoacyl-tRNA (methionine tRNA) with anitocodon 3’-UAC-5’ pair with the codon.
then Large ribosomal subunit joins the complex, completing the ribosome. tRNA is in the P site at this point.
- Elongation: Ribosome slides along the mRNA 5’->3’, adding new aminoacids as it goes. H bonds form bw the mRNA codon in the A site and the complementary tRNA anticodonl. This fills the A site. We now havea charged aminoacyl-tRNA in both A and P site.
Peptidyl transferase, uses the energy that was stored in the aminoacyl-tRNA complex to catalyze the fomration of a peptide bond. (look at diagram)
3.
Will mutations be inherited?
No, unless it is a mutation in the germ cell (sex)
Mutations are limited in the somatic cells.
DNA containing Viruses
Carry & location of transcription and translation
If they don’t carry any enzymes, will have to use hosts DNA and RNA polymerases in the nucleus.
If they bring DNA and RNA polymerases, they are the type that can not enter the nucleus where hosts DNA and RNA are restricted to.
RNA containing viruses
Carry and location of transcription and translation
- Bring RNA replicase: enzymes that replicate RNA into mRNA
- Don’t bring enzymes: their RNA is already in mRNA format
- Retrovirus:bring reverese transcriptase, RNA-> DNA, so host transcribes and translates. Only way to remove this virus is by lyseing the infected cell.
lytic vs lsogenic cycles
Bacteriophages
lytic: viral chromosome enter bacterial chromosome and produce progeny, with enough progency lyse the cell.
lsogenic: viral chromosome encorporated into bacterial chromsome and included in every bacterial division.
Transformation
integration of foreign chromosome fragment (plasmid) into the host genome.
Conjugation
bactieral form of sexual reproduction, 2 cells forming cytoplasmic bridge that allows fhr the transfer of genetic material. From donar male (+) to recipient female (-). Sex pili are found in donar male called F+, after they conjugate with female F-, female becomes F+ male.
Transduction
accidental method of genetic recombinati0on, bacteriophages intgrate into the host genome, they do not always perfectly excise. Bacterial genes will be packaged along with the viral genome. The virus then infects a new hosts bacterium and integrate the lysed bacterial chromosome.
Prok gene regulation
primarly at the transcription level
- Operon: directs RNA polymerase access to genome.
Operon consist of
- Structural genes: code for proteins of interest
- Operator site: nontranscribable region of DNA that is capable of binding a repressor protein
- Promoter site: nontranscribable region of DNA that RNA polymerase binds
- Operon function, and order
- structural gene function
- operator gene function
- promoter gene function
- regulator gene function
- directs RNA polymerase to genome
(regulator gene)- promoter gene - operator gene -(structural genes)
- transcribes protein
- nontranscribable DNA, region where repressor can bind
- nontrascribable DNA, reigion where RNA polymerase can bind
- transcription of regulator enzyme only
Gene Regulation of PROK
Inducible system
Normally, transcription is not occuring because of the presence of a repressor enzyme, which is binded to the operator. Becoming a road block for RNA polymerase at the promoter.
To induce transcription, inducer enzyme competes with the operator for the active site of the repressor enzyme.
If Inducer binds to the repressor’s active site, repressor can not bind to the operator, not blocking the RNA polymerase, and allowing transcription to occur.
Gene regulation for PROK
Repressible
Constant transcription occuring
Regulator gene, transcribes repressor that requires a corepressor.
It is only when corepressor and repressor enzyme form a complex that the repressor can carry out its function. To bind to the operator gene and road block the RNA polymerase path.
Enzymes / Indicate what process it aids and function
- Helicase
- Peptidyl Transferase
- Topoisomerase
- Release Factor
- RNA polymerase
- DNA polymerase III
- DNA polymerase II
- tRNA synthetase
- Repressor
- Inducer
- ssbp
- Primase
13: Ligase - Initiation factor
- Rep: unwinds double helix, creating single strand areas
- transL: forms peptide bond bw the amino acids, uses ATP
- Rep: relieve strain of the supercoiled DNA
- transL: when stop codon (UGA, UAA, UAG) coms up, instead of aminoacyl-tRNA, release factor binds at the A site. Causing a water molecule to bind to the polypeptide. And polypeptide chain will be released from the tRNA in the P site.
- transC: binds to promoter region and transcribes hnRNA
- rep: binds to the many primers, and replicates 5’->3’ (adding at the 3’ end) and also proofreads.
- rep: replaces primers with DNA in direction of 3’->5’. and also prrofreads.
- transL: uses GTP to bind amino acids to the tRNA forming aminoacy-tRNA complex.
- transC: binds to operator region and blocks RNA polyjmerase. (in inducer system of prok)
- transC: binds to repressor active site, and prevent repressor from binding to the operator. (in inducer system of prok)
- rep: holds single strands (antiparallel) apart
- rep: put down RNA primers
- rep: join okazaki framgents (3’->5’) by adding the phosphate backbone. (after DNA polymerase II added the nucelotides)
- transL: allows mRNA to bind to the small ribosome, (small ribosome then travels 5’->3’ untill it reaches AUG.)
Translation
(sites snd function)
(steps and energy usage)
A site: where new aminoacyl-tRNA bind
P site: where previous aminoacyl-tRNA moves, (aka translocation) when new aminoacyl-tRNA arrives at A site. (peptidyl transferase binds the amino acids of A and P site)
Initiation: binding formation of aminoacyl-tRNA req GTP
Elongation: Translocation, and peptidyl transferase forming peptide bonds uses stored GTP.
Termination: uses GTP to release polypep from tRNA and dismantel small and lg ribosomal subunits.
Translocation
term used in translation, where aminoacyl-tRNA goes from A site to P site.
\\\Mutations
- insertion
- deletion
- point mutation
- can lead to frameshift
- can lead to frameshift
- missense, nonsense, silent (change in one base pair)
nonsense
premature stop codon, (UGA, UAA, UAG)
point mutation
missense
change in the amino acid
(point mutation)
silent
due to degenerative genetic code, if 3rd nucleotide, most of the time the amino acid will not be changed. therefore no effect on phenotype.
(point mutation)
Lac operon vs Trp operon
Lac: inducer system (normally off)
Trp: repressible system (normally on)
