AP BIO Unit 6 - Molecular Genetics

How does DNA control a cell?

-by controlling protein synthesis

-proteins are the link between genotype and phenotype

Central Dogma of Gene Expression

-DNA genetic info transcribed into mRNA, then translated to protein

The Genetic Code

-sequences of bases in DNA correspond to the sequence of amino acids in proteins

-there are only 4 bases but 20 naturally occurring amino acids

- "codon" = three consecutive bases code for each amino acid

-one gene codes for one protein

Codon Dictionary

-Start = AUG

-Stop = UAA, UAG, UGA

-64 total combos

-each amino except "start" has multiple codons

-this helps safeguard against transcription (writing) or translation (reading) the code

Code Redundancy

-third base in a codon shows "wobble"

-first two bases are the most important in reading the code and giving the correct AA

-the third base often doesn't matter

Importance of Wobble

-allows for fewer types of tRNA

-allows some mistakes to code for the same AA which gives exactly the same polypeptide (silent mutation)

Reading Frame

-the "reading" of the code is every three bases

-EX. ATT GAT TAC ATT

UAA CUA AUG UAA

Stop Leu Start Stop

Code Evolution

-the genetic code is nearly universal

-Ex. CCG = proline (found in all life forms)

-reason the code must have evolved very early

-life on earth must share a common ancestor

-biotech applications use the universal nature of DNA to move genes from species to species

TRANSCRIPTION

-the DNA directed synthesis of RNA

-promoter = specific nucleotide sequence along the DNA

-in eukaryotes these promoters include a TATA box

-terminator = end of transcription (UAA, UAG, UGA)

STEP BY STEP TRANSCRIPTION

Initiation, Elongation, Termination

Initiation

-RNA polymerase binds to promoter

-this causes the DNA to unwind and separate

-RNA transcript begins to form

Elongation

-the template strand is transcribed by adding complementary RNA nucleotides in 5' - 3' direction

-the RNA transcript grows & DNA reforms double helix

-in prokaryotes, the transcript is immediately usable as mRNA

Termination

-once a termination sequence is transcribed, the RNA transcript is released & RNA polymerase detaches from DNA

-there are three termination codes - UAA, UAG & UGA

RNA Modification in Eukaryotic Cells

- 5' cap & poly-A tail are added to RNA transcript

-prevents breakdown of DNA

-facilitates passage though nuclear pores

-cap provides point of attachment at ribosome

RNA Processing - RNA Splicing

-Introns = non-coding segments of eukaryotic genes

-Exons = coding segments

-During RNA processing introns are removed and exons are spliced together

-Splicesomes = an assembly of snRNPS (ribonuclear proteins) & other proteins bind to the end of introns; recognize code sequence

-newly spliced exon; only mRNA can exit nucleus

Functional & Evolutionary Importance of Introns

-introns may play regulatory roles in the cell

-benefit o split genes is the to enable a single gene to encode more than one kid of protein

-facilitates the shuffling among genes promoting evolution

Alternative Splicing

-the RNA can be splice into diff. mRNA's

-each diff, mRNA produces a diff. polypeptide

-this explains how the body can create infinite # of antibodies

-Ex. Bcl-XL and DSCAM gene

Commentary

-about 60% of genes are estimated to have alternative splicing sites

-one gene does not equal one polypeptide

PROTEIN SYNTHESIS

-translation is the RNA directed synthesis of a protein

-one codon = one amino acid

-transfer RNA (tRNA) molecules interpret the genetic code as written on the mRNA transcript

Role of Transfer RNA

-links mRNA codon to its matching amino acid

- "anticodon" = complementary base sequence to mRNA codon

-amino acid attachment site is located opposite the anticodon

Ribosomes

-Structure = large & small subunit both composed of protein & rRNA

-one enzyme for each amino acid

-Facilitate = codon-anticodon complex formation; peptide bond formation

-prokaryotic ribosomes are often the target of antibiotics

Recognition steps for accurate translation

1.) correct match between the tRNA & the amino acid; each amino acid has a specific enzyme that aids in attachment to tRNA (aminoacy-tRNA synthetase)

2.) codon - anticodon bonding insures translation

INITIATION of Translation

-mRNA binds to a small ribosomal subunit, AUG is the "start" sequence

-GTP provides energy needed to bring the large subunit to create a complex

-E = exit; P = protein building site; A = enzyme driven amino acid binding site

ELONGATION of Polypeptide Chain

1.) codon recognition

2.) rRNA serves as a ribozyme- catalyst of peptide bond

3.) translocation- ribosome shifts the mRNA by one codon- both move relative to each other

TERMINATION

-stop codon reaches "A" site

-release factor matches stop codon & protein is released

-ribosome complex disintegrates

Free vs Bound Ribosomes

-Free ribosomes = protein is needed in cytosl

-Bound ribosomes = protein is needed at membrane or beyond via ER; true of cells of secretory organs or tissues

-signal recognition particle carries the complex to the ER membrane = signal recognition protein temporarily binds ribosome to the ER membrane; protein is fed into cisterna as it elongates

Not all info flows in the same direction...

Viruses vs Prokaryotic cells

Viruses

-nucleic acid core is either expressed by HOST cell OR nucleic acid joins host DNA to be expressed later

-Pro = uses host to live, not ER or golgi app.

-Con = totally dependent on host

Prokaryotic Cells

-transcription is immediately followed by translation

-Pro = quick, fast process

Retroviruses

-genome is RNA

-genetic info flows backwards or in reverse direction

-reverse transcriptase in virus = RNA to DNA flow

-newly made DNA = provirus never leaves host cell

- DNA to RNA which can make viral proteins or genome for new virus particles

Viral reproductive cycle

-only reproduce within a HOST cell

-HOST RANGE = specific host cell per virus type (broad or narrow range)

-host specifically is like "lock & key" analogy, uses surface proteins

Protein Synthesis in Bacterium/ Prokaryotic Cell

-no nucleus, so mRNA is translated right after transcription

-no introns or exons; bacteria have streamlined DNA; little to no "junk"

-ribosomes are smaller, differ in chemical makeup from eukaryotic cell

Mutations

-changes in DNA code sequence

-chromosomal mutations = large scale changes to chromosome structure

-point mutations = small scale changes in just one base pair of a gene (substitution, insertion, deletion)

Ch. 15 - The Regulation & Control of Gene Expression

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Why would cells regulate gene expression?

-conservation of resources = express genes when proteins are needed

-organization = control of processes based on environmental conditions (ex. metabolism)

-coordination = express genes in a timely sequence (ex. development)

Expression of genes - starts in nucleus

-occurs in transcription

-regulatory proteins attach to promoter (transcription factors)

-RNA polymerase attaches & builds mRNA

Control of Gene Expression in Eukaryotic Cells

-complicated process

-main control levels = 1.) nucleus- those inside the nuclear membrane 2.) cytoplasm- those that occur outside the nucleus

-transcription initiation is the most important control point for ALL life forms

1.) Regulation of chromatin structure

-heterochromatin = partially coiled; not available for transcription; visible w/ a light microscope

-euchromatin = uncoiled; available for transcription

2.) Chromatin modification - in nucleus

- DNA methylation = attachment of methyl groups to cytosine; blocks transcription (effect); genomic imprinting-expression of either maternal or paternal allele

-Histine acetylation - attachment of aceytl groups to histones; loosens grip of histines on DNA-enables transcription (effect)

3.) Transcriptional Regulation (Eukaryotic Cells)

-introns and exons

-control elements noncoding DNA that help regulate transcription of gene by binding to proteins like promoters (ex. TATA box)

Gene Regulation in Nucleus

-promoter = noncoding region of gene that controls its expression, located next to the gene they regulate

-enhancer = mechanism in promoter region that acts as a "switch"; enables gene to be turned "on" or "off"

Transcription Factors

-proteins that amplify or reduce gene expression

4-7.) Post-Transcriptional Regulation in the Cytoplasm

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4.) Alternative Splicing

-different m RNA molecules are produced from the same primary transcript; depends upon which RNA segments are treated as exons or introns: RESULT = more than one protein from a single gene

5.) RNA degradation

-mRNA is long lived in eukaryotes so not as adaptable to environ changes like prokaryotes

6.) Translation blockage

-regulatory proteins bind to leader region of 5' end of mRNA; ribosomes can't attach

7.) Protein degradation

- processing of protein or chemical modifications to male a protein functional; occurs in "transport"

-selective degradation = cells way of limiting the lifetime use of certain proteins

Operon Model

-form of gene control in bacteria

-Jacob & Monod = prokaryotic model of gene control

-bacteria have limited DNA = efficient structure & use of info.

-operons = cluster of functionally related genes

-Advantage = single "on-off" switch controls the cluster

-repressible or inducible operons

Operon Structure

1.) Regulatory Gene: makes repressor protein which may bind to the operator

2.) Operon Area: promoter = attachment sequence on the DNA for RNA polymerase; operator = binding site for the repressor protein; structural genes = makes the enzymes for the metabolic pathway

Lac Operon

- for digesting lactose

-Inducible Operon = only works ("on") when the substrate (lactose) is present

-if lactose is absent operon is "off", no transcription occurs, no enzymes made

-lactose absent = repressor protein binds to operator tubes blocking RNA polymerase

-lactose present = isomer of lactose blocks or binds repressor; RNA polymerase can bind & transcribe 3 genes needed to digest lactose

trp Operon - Synthesis of Tryptophan

-Repressible Operon = only works "on" when substrate (tryptophan) is absent

-examples of feedback inhibition

-result = maintain constant level of tryptophan

-tryptophan absent = repressor protein is inactive, operon "on" tryptophan made; "Normal" state for the cell

-tryptophan present = repressor protein is active, operon "off", no transcription, no enzymes; no tryptophan made (result)

Genetics of Bacteria

-bacteria have a ROUND DNA molecule as well as a plasmid (smaller # of genes)

-bacterial cells divide by binary fission

-DNA replication occurs rapidly from a single point

Transduction - generalized

-virus carries gene from host to host

-piece of bacterial DNA is inserted into a viral capsid

Transduction - specialized

-temperate virus incorporates its DNA into a host's genome to form a prophage

-prophage DNA leaves taking hos DNA with it

-the phage takes the host gene into next host; recombination occurs

Conjugation

-donation of plasmid copy occurs though pilus; ONE WAY

-cytoplasmic bridge forms as bacteria draw near

-F factor (fertility genes) must be present; genes for making pilus

-F+ = male / F = female

Transformation

-absorption of DNA from environment

-proteins on bacteria cell surface are specialized for uptake of naked DNA from surroundings

-some bacteria can be shocked into taking up DNA

Recombination & Transformation

-how do scientists account for introns when using bacteria cells as factories? = create cDNA (complementary DNA) using fully processed mRNA copy of the eukaryotic gene in vitro along with reverse transcriptase; genes in bacteria are easier to clone

How do you know you have the gene in question?

-nucleic acid probes = radioactively or florescently labeled single stranded DNA or RNA which "base pairs" w/ complementary sequence of DNA or RNA

-USE: locate cloned genes in bacterial culture, identify bands or gels, diagnose infectious disease

Polymerase Chain Reaction

-in vitro (w/out cells) = procedure done in a test tube or petri dish

-USE: rapidly produce multiple copies of a genes or a select length of DNA no matter how small

What is done with all this info?

-Genomic libraries = collection of bacterial or phage clones

-USE: source for other genes of interest; or gene mapping

How do scientists know they'll get the right fragments?

-precloning fragment selection = PCR; Electrophoresis

-postcloning fragment selection = differential media; Hybridization

Gene Cloning

-production of multiple copies of a gene (human genes or any eukaryotic gene) using a bacterial plasmid

-2 main steps used to create plasmid = isolation of gene; recombination & transformation

Post Cloning - Differential Media

-culture bacteria on medium that would only allow survival of cells w/ desired gene

-Ex. agar w/ ampicillin & agar w.out

Post Cloning - Hybridization

-gene clone is denatured (split)

-radioactive probes are added

-colonies that hydrogen bond w/ probes are identified via fluorescence

-hybridized colonies from original plate are cultured

DNA Fingerprinting

-Precloning fragment selection = gel-electrophoresis separation of molecules based on size; negatively charged DNA molecules are pulled through gel by an electrical field; smaller molecules travel faster & farther

Restriction Fragment Length Polymorphism

-RFLP's are used to distinguih DNA from homologous segments

-inherited in a mendelian fashion = genetic marker for mapping chromosomes; useful in pedigree studies

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Each DNA strand has two unique ends. The 3' end has a hydroxyl (-OH) group on the deoxyribose sugar, whereas the 5' end has a phosphate group. In the double helix, the two strands are antiparallel, that is, they run in opposite directions such that the 3' end of one strand is adjacent to the 5' end of the other strand.

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DNA polymerase III can only add nucleotides to the 3' end of a new DNA strand. Because the two parental DNA strands of a double helix are antiparallel (go from 3' to 5' in opposite directions), the direction that DNA pol III moves on each strand emerging from a single replication fork must also be opposite.

For example, in the replication fork on the left, the new strand on top is being synthesized from 5' to 3', and therefore DNA pol III moves away from the replication fork. Similarly, the new strand on the bottom of that same replication fork is being synthesized from 5' to 3'. But because the bottom parental strand is running in the opposite direction of the top parental strand, DNA pol III moves toward the replication fork.

In summary, at a single replication fork, one strand is synthesized away from the replication fork, and one strand is synthesized toward the replication fork. When you look at both replication forks, note that a single new strand is built in the same direction on both sides of the replication bubble.

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At each replication fork, helicase moves along the parental DNA, separating the two strands by breaking the hydrogen bonds between the base pairs. (This makes the two parental DNA strands available to the DNA polymerases for replication.) As soon as the base pairs separate at the replication fork, single-strand binding proteins attach to the separated strands and prevent the parental strands from rejoining.

As helicase separates the two parental strands, the parental DNA ahead of the replication fork becomes more tightly coiled. To relieve strain ahead of the replication fork, topoisomerase breaks a covalent bond in the sugar-phosphate backbone of one of the two parental strands. Breaking this bond allows the DNA to swivel around the corresponding bond in the other strand and relieves the strain caused by the unwinding of the DNA at the helicase.

Short segments of newly synthesized DNA are joined into a continuous strand by _____.

ligase

After DNA replication is completed, _____.

each new DNA double helix consists of one old DNA strand and one new DNA strand

The first step in the replication of DNA is catalyzed by _____.

helicase

The action of helicase creates _____.

replication forks and replication bubbles

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Since DNA polymerase can assemble DNA only in the 5' to 3' direction, the new strand complementary to the 3' to 5' strand must be assembled either in short 5' to 3' segments, which are later joined together by ligase, or be assembled continuously.

The synthesis of a new strand begins with the synthesis of a(n) _____.

RNA primer complementary to a preexisting DNA strand

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Primase catalyzes the formation of an RNA primer.

An old DNA strand is used as a _____ for the assembly of a new DNA strand.

template

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Nucleotides are composed of a pentose sugar, a nitrogenous base, and a phosphate group.

You can tell that this is an image of a DNA nucleotide and not an RNA nucleotide because you see a _____.

sugar with two, and not three, oxygen atoms

Purines

adenine & guanine; double-ring structures

Pyrimidines

thymine, cytosine & uracil; single-ring structures

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The nitrogenous base is attached to the sugar's 1' carbon and the phosphate group is attached to the sugar's 5' carbon.

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New nucleotides are added to the 3' end of a growing polynucleotide

Where does translation take place?

ribosome

Which nucleic acid is translated to make a protein?

mRNA

Which of the following processes is an example of a post-translational modification?

phosphorylation; enzymes can phosphorylate proteins to alter their activity.

Which of the following steps occurs last in the initiation phase of translation?

The large ribosomal subunit joins the complex; This step occurs after the 5' mRNA is bound by the ribosome and the start codon is bound by an aminoacyl tRNA.

At which site do new aminoacyl tRNAs enter the ribosome during elongation?

A-site

What is meant by translocation?

The ribosome slides one codon down the mRNA.

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There are no tRNAs complementary to the three stop codons; termination occurs when release factors recognize the stop codon in the A-site and catalyze the release of the polypeptide from the tRNA in the P-site.

What enzyme catalyzes the attachment of an amino acid to tRNA?

aminoacyl-tRNA synthetase; This enzyme matches a particular tRNA with a particular amino acid.

The initiator tRNA attaches at the ribosome's _____ site.

P site

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A codon is a group of three bases that can specify only one amino acid.

_____ bind(s) to DNA enhancer regions.

activators

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The bending of the DNA allows for the interaction of transcription factors and RNA polymerase.

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Both RNA polymerase and transcription factors bind with the promoter.

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Transcription is the process by which information encoded in DNA is converted to information encoded in RNA.

Control of Gene Expression

1) unpacking of DNA 2) transcription of gene 3) processing of RNA 4) export of mRNA 5) breakdown of mRNA 6) translation of mRNA 7) breakdown of protein 8) activation of protein

TRANSCRIPTION is the #1 control point of gene expression

The operon model of the regulation of gene expression in bacteria was proposed by _____.

Jacob & Monod

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Transcription is inhibited when a regulatory protein binds to the lac operon operator.