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nucleotides
basic subunits of RNA or DNA consisting of purine or pyrimidine covalently bonded to ribose or deoxyribose
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nucleobase
- also simply called a base
- 4 different ones for DNA:
- Adenine
- Thymine
- Cytosine
- Guanine
- *Set of 3 nucleotides encodes a specific amino acid, string of amino acids make up protein
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genome
- a complete set of genetic info of a cell
- includes plasmids as well as the chromosomes; however genome is often used interchangeably with chromosome
- *The genome of all cells is composed of DNA, but some viruses have an RNA genome
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gene
- the functional unit of the genome
- encodes a product, most commonly a protein
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genomics
the study and analysis of nucleotide sequence of DNA
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2 general tasks all cells must accomplish before multiplying
- 1. the double-stranded DNA must be duplicated
- 2. info encoded by DNA must be decoded so cell can synthesize necessary gene products
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DNA replication
duplication of DNA so encoded information can be passed to next generation
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gene expression
- decoding information which is encoded by DNA so that the cell can synthesize the necessary gene products
- Involves two interrelated events: Transcription and translation
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Transcription
- refers to making the RNA molecule
- Process of copying the info encoded in DNA into RNA
- Involves Initiation, Elongation, and Termination
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Translation
interprets info carried by mRNA to synthesize the encoded protein
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Central dogma of molecular biology
refers to the flow of info from DNA to RNA to protein
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How are the nucleotides of DNA bonded together
- Joined together by covalent bond between the 5'PO4 (5 prime phosphate) of one nucleotide and the 3'OH (3 prime hydroxyl) of the next
- *5' and 3' refer to numbered carbon atoms on pentose sugar of nucleotide
- *The nucleotides joining in the manner creates the sugar-phosphate backbone
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Explain the ends of DNA strands
- Because of the chemical structure of nucleotides and how they join together, a single strand of DNA will always have a 5'PO4 at one end and a 3'OH at the other
- Often referred to as the 5' end (5 prime end) and the 3' (3 prime end)
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What bonds occur between the nucleobases?
- The two stands of DNA are complementary and are held together by hydrogen bonds
- Amy marries Tim (with 2 hydrogen bonds)
- Guenevere marries Chris (with 3 hydrogen bonds)
*Because Amy and Tim only have 2 bonds, Amy steps out with Urical with RNA is around. Bond isn't a tight... marriage sucks!
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base pairing
- refers to the characteristic bonding of A to T, and G to C
- Is the fundamental characteristic of DNA
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antiparallel
- refers to the double helix in DNA and the two strands being complementary
- They are also antiparallel, meaning they oriented in opposite directions
- One strand is oriented in the 5' to 3' direction, it's complement is oriented in the 3' to 5' direction
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How is RNA synthesized?
Using a region of ONE of the two strands of DNA as a template
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3 different functional types of RNA, required for gene expression
- All transcribed from different sets of genes:
- mRNA (messenger RNA)
- rRNA (ribosomal RNA)tRNA (transfer RNA)
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mRNA
- messenger RNA
- info encrypted is deciphered according to genetic code, which correlates each set of 3 nucleotides to a particular amino acid
- *Some genes are never translated into proteins, as the RNAs themselves are final products
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How does a cell regulate gene expression
- fundamental aspect of gene regulation is instability of mRNA
- Within minutes of being produced, transcript are degraded by enzymes
- If gene if turned on, will continue to be available to translation.
- If turned off, # of transcripts will rapidly decline
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Describe DNA replication
- Process is generally bidirectional, meaning it proceeds in both directions from the starting point.
- This allows a chromosome to be replicated in half the time it would take if the process were unidirectional
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replication forks
- *The progression of bidirectional replication around a circular DNA molecule creates two advancing forks where DNA synthesis is occurring
- The replicating forks ultimately meet at a terminating site when process is complete
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semiconservative
- refers to half the original molecule being conserved in each new molecule when DNA is replicated
- The two DNA molecules created through replication each contain one of the original strands
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How is DNA replication initiated
Specific proteins must recognize and bind to origin of replication
Prokaryotic chromosomes and plasmids typically contain only one of these initiating sites - Proteins bind to site, cause localized melting of double-stranded DNA, exposing single-stranded regions for templates
- Primases (enzymes) then synthesize short stretches of RNA
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origin of replication
distinct region of DNA molecule at which replication is initiated
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Primers
- Small fragments of nucleic acid to which DNA polymerase can add nucleotides
- (the enzyme can add nucleotides only to an existing fragment)
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Replisome
The complex of enzymes and other proteins that synthesize DNA
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DNA gyrase
Enzyme that temporarily breaks the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix
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DNA ligase
Enzyme that joins two DNA fragments by forming a covalent bond btwn the sugar and phosphate residues of adjacent nucleotides
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Primase
Enzyme that synthesizes small fragments of RNA to serve as primers for DNA synthesis
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DNA polymerases
- Enzymes that synthesize DNA
- use one strand of DNA as template to make the complementary strand
- Nucleotides can be added only to the 3' end of an existing fragment - therefore, synthesis always occurs in the 5' to 3' direction
- *Cannot initiate synthesis
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Helicases
Enzymes that unwind the DNA helix ahead of the replication fork
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Leading strand
The DNA strand that is synthesized as a continuous fragment, as the DNA polymerase simply adds nucleotides to the 3' end
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Lagging strand
- The DNA strand that is synthesized in a series of fragments
- More complicated than leading strand as DNA polymerases can't add nucleotides to 5' end, so as additional template is exposed synthesis must be reinitiated
- Each time synthesis is reinitiated, another RNA primer must be made first.
- This results in the small fragments, each of which has short stretch of RNA at 5' end.
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Okazaki fragments
Name of small nucleic acid fragments made during synthesis of lagging strand of DNA
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RNA polymerase
- in transcription, enzyme that synthesizes single-stranded RNA molecules from a single-stranded DNA template
- Like DNA polymerase, RNA polymerase can add nucleotides ONLY to 3' end and therefore ONLY synthesizes RNA in 5' to 3' direction
- UNLIKE DNA polymerase, RNA polymerase can start synthesis w/o a primer
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Promoter
- Nucleotide sequence to which RNA polymerase binds to initiate transcription
- ID's regions that will be transcribed
- Also orient the direction of RNA polymerase on DNA molecule, dictating which strand will be used as template
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Terminator
- Nucleotide sequence at which RNA synthesis stops
- RNA polymerase falls off the DNA template and releases the newly synthesised RNA
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minus ( - ) strand
- The DNA strand that serves as the template for transcription
- Resulting RNA molecule is complementary to this strand
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plus ( + ) strand
- Strand of DNA complementary to the one that serves as the template for RNA synthesis
- the nucleotide sequence of the RNA molecule is the same as this strand, except it has uracil rather than thymine
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Monocistronic vs Polycistronic
- *In prokaryotes, mRNA can carry info for one or multiple genes
- Monocistronic is a transcript that carries one gene
- Polycistronic carries multiple genes
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Sigma (σ ) factor
- Portion of RNA polymerase that recognizes the promoter regions
- A cell can have different types of σ factors that recognize different promoters, allowing the cell to transcribe specialized sets of genes as needed
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What occurs in elongation phase of RNA transcript
- RNA polymerase moves along DNA, using minus strand as template to synthesize single-stranded RNA molecule
- Nucleotides are added only to 3' end
- Reactions fueled by hydrolyzing high energy Phosphate bonds of incoming nucleotide
- When RNA polymerase advances, denatures new stretch of DNA & allows previous section of close
- This exposes new region of template so elongation can continue
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During translation, how is information deciphered
From mRNA, using the genetic code, which correlates a series of 3 nucleotides, a codon, with one amino acid
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codon
- Set of 3 adjacent nucleotides that encode either an amino acid or the termination of the polypeptide
- Because of triplet combination btwn 4 different nucleotides, there are 64 different codons
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reading frames
- ways in which triplets can be grouped
- Each 2 letter group
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What is the "start" reading frame
AUG
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What are the "stop" reading frames
- UAA
- UAG
- UGA
- "University of AlabamA"
- "U
niversity of Alabama and Georgia" - "University of Georgia and Alabama"
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Ribosomes
- Structure that serve as translation machines
- facilitates the joining of amino acids during the process of translation
- does so by aligning two amino acids so ribosomal enzyme can easily create peptide bond btwn them
- composed of protein and ribosomal RNA
- The prokaryotic ribosome (70S) consists of a 30S and 50S subunit
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tRNA
- segments of RNA which act as keys to genetic code
- Recognizes and base-pairs with certain codons and, in the process, delivers the appropriate amino acid to that site
- Recognition is possible cause each tRNA has an anticodon
- Once it's donated it's amino acid during translation, can be recycled
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anticodon
- sequence of 3 nucleotides in a tRNA molecule that is complementary to a particular codon in mRNA
- Allows tRNA to recognize and bind to appropriate codon
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ribosome-binding site
- Sequence of nucleotides in mRNA to which a ribosome binds
- the first time the codon for methionine (AUG) appears after that site, translation generally begins
*AUGustus MaCrae gets me going"
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start codon
- Codon at which translation is initiated
- typically the first AUG after a ribosome-binding site
- *AUG only functions as start codon when preceded by ribosome-binding site
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Stop codon
- Codon that terminates translation, signaling the end of the protein
- 3 stop codons
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Termination of translation
- Elongation is terminated when ribosome reaches a stop codon, which is a codon not recognized by tRNA
- Enzymes free polypeptide by breaking covalent bond that joins it to RNA
- Ribosome falls off mRNA, dissociating into it's 2 subunits (30S & 50S) which can then be reused to initiate translation at other sites
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chaperones
proteins that assists folding polypeptides into specific shapes
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singal sequence
- characteristic series of hydrophobic amino acids at their amino terminal end, which "tags" polypeptides destined for transport
- must be removed by proteins in the membrane
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pre-mRNA
- specific to eukaryotes
- must be processed (formed) both during and after transcription to form mature mRNA
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Major differences specific to Prokaryotes in Transcription and Translation
- mRNA is NOT processed
- mRNA does not contain introns
- Translation of mRNA begins as it is being transcribed
- mRNA is often polycistronic; translation usually begins at the first AUG codon that follows a ribosome-binding site
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Major differences specific to Eukaryotes in Transcription and Translation
- A cap is added to the 5' end of mRNA, and a poly A tail is added to the 3' end
- mRNA contains introns, which are removed by splicing
- the mRNA transcript is transported out of the nucleus to that it can be translated in the cytoplasm
- mRNA is monocistronic, translation begins at the first AUG
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signal transduction
- transmits info from outside a cell to the inside
- allows cells to monitor and react to environmental conditions
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quorum sensing
- a phenomenon in which some organisms can "sense" the density of cells within their own population
- Allows cells to activate genes that are only useful when expressed by a critical mass
- Involves the bacteria "talking" to each other
- Only when a signaling molecule reaches a critical level does it induce the expression of specific genes
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two-component regulatory system
- an important mechanism cells use to detect and react to changes in the external environment
- consist of 2 different proteins - a sensor and a response regulator
- Sensor spans cytoplasmic membrane. In response to enviro variation, sensor chemically modifies a region on it's internal portion. This alerts the response regulator which can turn genes on or off, depending on the system
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antigenic variation
- an alteration in the characteristic of certain surface proteins
- Pathogens that do this can stay one step ahead of body's defenses by altering the very molecules our immune systems learn to recognize
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phase variation
- another mechanism of randomly altering gene expression
- the routine switching on and off of certain genes
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operon
a set of regulated genes transcribed as a single polycistronic message
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regulon
- separate operons controlled by a single regulatory mechanism
- two component regulatory systems often control regulons
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global control
simultaneous regulation of numerous genes
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when describing enzymes, how do scientists group them?
- According to the type of regulation that governs their synthesis:
- Constitutive
- Inducible
- Repressible
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Constitutive
- Enzymes are synthesized constantly
- genes that encode these are always active
- Usually play indispensable roles in central metabolic pathways
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Inducible
- enzymes that are not routinely produced at significant levels; instead, their synthesis can be turned on when needed
- Often involved in the transport and breakdown of specific energy sources
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Repressible
- Enzymes are produced routinely, but their synthesis can be turned off when they are not required
- Generally involved in biosynthetic (anabolic) pathways
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What are the two most common regulatory mechanisms to control transcription
- Alternative sigma factors
- DNA-binding proteins
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Alternative sigma factors
Recognize different sets of promoters, thereby controlling the expression of specific groups of genes
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DNA-binding proteins
- *transcription is often controlled by proteins that bind to specific DNA sequences
- When a regulatory protein attaches to DNA, it can act either as a repressor, which blocks transcription, or an activator, which facilitates transcription
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Repressor
- a regulatory protein that blocks transcription (negative regulation)
- Does this by binding to operator, a specific DNA sequence located immed downstream of a promoter
- Makes it so RNA polymerase cannot progress past
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What 2 different mechanisms do repressors function:
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Induction
- The repressor is synthesized as a form that binds to operator, blocking transcription.
- A molecule called an inducer attaches to repressor, changes shape of repressor so it no longer grasp operator
- With repressor unable to bind DNA, RNA polymerase may transcribe the gene
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Repression
- The repressor is synthesized as form that cannot bind to operator
- When a molecule termed a corepressor attaches to repressor, the corepressor-repressor complex can then bind to operator, blocking transcription
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Activators
- A regulatory protein that facilitates transcription (pos regulation)
- Genes controlled by activators have ineffective promoter preceded by an activator-binding site
- Binding of activator to DNA enhances ability of RNA polymerase to initiate transcription at that promoter
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Activator-binding site
Nucleotide sequence that precedes an ineffective promoter
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lac operon
- Encodes proteins involved with the tranport and degradation of lactose, and is only turned on when lactose in in the cell but glucose is not available
- Uses a repressor that prevents transcription when lactose is not available; the repressor binds to operator, blocking RNA polymerase
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carbon catabolite repression
- CCR
- The phenomenon that when glucose is available, the lac operon is not expressed
- Global control system that allows glucose to regulate expression of the lac operon as well as other sets of genes
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diauxic growth
- the characteristic 2-phase growth pattern that can be seen when cells are using glucose for fuel
- When glucose is used up, growth stops for short period as cells gear up to begin metabolizing lactose, then cells begin multiplying again using lactose to fuel growth
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CAP
- catabolite activator protein
- one mechanism of carbon catabolite repression
- required for transcription
- must be bound by an inducer to work - an ATP derivative called cAMP
- inducer only made when extracellular glucose levels are low, as enzyme required for it's synthesis is activated by idle form of glucose transporter component
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inducer exclusion
- another mechanism of regulation
- When glucose is being moved into the cell, a glucose transport component binds to the lactose transporter, locking it in a non-functional position
- Can't move lactose into cell, so lac operon will not be induced
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RNA interference
- (RNAi)
- Cells use this routinely to destroy specific RNA transcripts
- Cell synthesizes short single-stranded RNA pieces to locate specific RNA transcripts destined for destruction
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bioinformatics
a new field which creates the computer technology to store, retrieve, and analyze nucleotide sequence data
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open reading frames
- ORF's
- protein-encoding regions in DNA which computers help locate
- stretches of nucleotide sequences generally longer than 300 bp that bein with a start codon and end with a stop codon
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Metagenomics
- the analysis of total microbial genomes in an environment
- By examining, researchers can study all microorganisms and viruses in a community, instead of just the relatively few that grow in a culture
- Also being used to study microbial life in the open oceans and in soils
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