AP Biology | Unit 6: Gene Expression and Regulation

Topic 1: DNA and RNA Structure

Discovery of DNA Structure

• In the 1950’s Rosalind Franklin performed X-Ray crystallography of DNA. Her work revealed a pattern that was regular and repetitive.

• During the same time, Edward Chargaff analyzed DNA samples from different species. He found the following rule held true for all species. The amount of adenine equals the amount of thymine and the amount of cytosine equals the amount of guanine.

• Watson and Crick combined the findings of Franklin (helix shape) and Chargaff (base pairing) to create the first 3D, double helix model of DNA

Nucleotide Structure

• Purines: Double ring structure that includes adenine and guanine

• Pyrimidines: Single ring structure that includes cytosine, uracil, thymine

Nucleotide Pairing

• base pairs are held together by hydrogen bonds

• adenine and thymine have two hydrogen bonds

• cytosine and guanine have three hydrogen bonds

Key Features of DNA Structure

• DNA is a double stranded helix that are made of a sugar-phosphate backbone and a nucleotide pairing center

• DNA strands are antiparallel where one strand runs in the 5 prime to 3 prime, while the other strand runs in opposite, upside-down direction 3 prime to 5 prime

• the 5 prime end has a free phosphate group

• the 3 prime end has a free hydroxyl group

Key Function of DNA

• DNA is the primary source of heritable information

• Genetic information is stored in and passed from one generation to the next through DNA

• the exception to this is RNA as it is the primary source of heritable information in some viruses

Prokaryotic vs Eukaryotic DNA

• Eukaryotic cells have the DNA found in the nucleus and have linear chromosomes

• Prokaryotic cells have DNA is in the nucleoid region and Chromosomes are circular

• prokaryotes (and some eukaryotes) also contain plasmids which are small, circular DNA molecules that are separate from the chromosomes

Plasmids

• Plasmids replicate independently from the chromosomal DNA

• plasmids are primarily found in prokaryotes

• plasmids contains genes that may be useful to the prokaryote when it is in a particular environment, but may not be required for survival

• plasmids can be manipulated in laboratories

• plasmids can be removed from bacteria, then a gene of interest can be inserted into the plasmid to form recombinant plasmid DNA

• when the recombinant plasmid is inserted back into the bacteria the gene will be expressed

• Bacteria can exchange genes found on plasmids with neighboring bacteria

• once DNA is exchanged, the bacteria cna express the genes acquired

• Helps with survival of prokaryotes

RNA vs DNA

• RNA: ribonucleic acid, single stranded, A=U and C=G

• DNA: deoxyribonucleic acid, double stranded, A=T and C=G

Topic 2: Replication

DNA Replication

• DNA replicates during the S phase of the cell cycle

Models of DNA Replication

• there are 3 alternative models for DNA replication: conservative, semi-conservative, dispersive

• conservative model: the parental strands direct synthesis of an entirely new double stranded molecule. the parental strands are fully “conserved”

• semi-conservative model: two parental strands each make a copy of itself and after one round of replication, the two daughter molecules each have one parental and one new strand

• dispersive model: the material in the two parental strands in dispersed randomly between the two daughter molecules and after one round of replication, the daughter molecules contain a random mix of parental and new DNA

Which Model Is Correct?

• In 1954 Meselson and Stahl performed an experiment using bacteria

• the process: bacteria was cultured with a heavy isotope, and is N^15. bacteria was transferred to a medium with N^14, a light isotope. Dna was centrifuged and analyzed after each replication

• by analyzing samples of DNA after each generation, it was found that the parental strands were following the semi-conservative model

Steps in DNA Replication

1) DNA replication begins at sites called origins of replication

• various proteins attach to the origin of replication and open the DNA to form a replication fork

  2) helicase will unwind the DNA strands at each replication fork

• to keep the DNA from re-bonding with itself, proteins called single strand binding proteins (SSBPs) bind to the DNA to keep it open

• Topoisomerasee will help prevent strain ahead of the replication fork by relaxing supercoiling

  3) the enzyme primase initiates replication by adding short segments of RNA, called primers, the other parental DNA strand

• the enzymes that synthesize DNA can only attach new DNA nucleotides to an existing strand of nucleotides

• Primers serve as the foundation for DNA synthesis

  4) antiparallel elongation: DNA polymerase 3 (DNAP III) attaches to each primer on the parental strand and moves in the 3 prime to 5 prime direction

• as it moves, it adds nucleotides to the new strand in the 5 prime to 3 prime direction

• the DNAP III that follows helicase is known as the leading strand and it only requires one primer

• the DNAP III on the other parental strand that moves away from helicase is known as the lagging strand and requires many primers

  5) the leading strand is synthesized in one continuous segment, but since the lagging strand moves away from the replication fork it is synthesized in chunks

• okasaki fragments: segments of the lagging strand

6) after DNAP III forms an Okazaki grament, DNAP I replaces RNA nucleotides with DNA nucleotides

• DNA ligase: joins the Okazaki fragments forming a continuous DNA strand

Problems at the 5’ End

• since DNAP III can only add nucleotides to a 3 prime end, there is no way to finish replication on the 5 prime end of a lagging strand

• over many replications this would mean that the DNA would become shorter and shorter

• Telomeres: repeating units of short nucleotide sequences that do not code for genes

• form a cap at the end of DNA to help postpone erosion

• the enzyme telomerase adds telomeres to DNA

Proofreading and Repair

• as DNA polymerase adds nucleotides to the new DNA strand, it proofreads the bases added

• if errors still occur, mismatch repair will take place

• enzymes remove and replace the incorrectly paired nucleotide

• if segments of DNA are damaged, nuclease can remove segments of nucleotides and DNA polymerase and ligase can replace the segments

Topic 3: Transcription and RNA Processing

Proteins

• Proteins are polypeptides made up of amino acids

• amino acids are linked by peptide bonds

• gene expression: the process by which DNA directs the synthesis of proteins

• this includes the two stages: transcription and translation which occurs in all organisms

Transcription and Translation

Transcription: the synthesis of RNA using information from DNA

• allows for the “message” of the DNA to be transcribed

• occurs in the nucleus

Translation: the synthesis of a polypeptide using information from RNA

• occurs at the ribosome

• a nucleotide sequence becomes an amino acid sequence

Types of RNA

• as we go through transcription and translation there will be three key RNA molecules

1) Messenger RNA (mRNA) carries info

2) Ribosomal RNA (rRNA) helps build ribosomes

3) Transfer RNA (tRNA) translates to sequence

Messenger RNA

• Messenger RNA is synthesized during transcription using a DNA template

• mRNA carries information from the DNA (at the nucleus) to the ribosomes in the cytoplasm

Transfer RNA

• transfer RNA molecules are important in the process of translation

• each tRNA can carry a specific amino acid

• can attach to mRNA via their anticodon

• a complementary codon to mRNA

• allow information to be translated into a peptide sequence

Ribosomal RNA

• rRNA helps form ribosomes

• helps link amino acids together

The Genetic Code

• DNA contains the sequence of nucleotides that codes for proteins

• the sequence is read in groups of three called the triplet code

• during transcription, only one DNA strand is being transcribed

• known as the template strand (also known as the noncoding strand, minus strand, or antisense strand)

• mRNA molecules formed are antiparallel and complementary to the DNA nucleotides

• mRNA nucleotide triplets are called codons

• codons code for amino acids

• there are 64 differents codon combinations where 61 code for amino acids and 3 are stop codons

• universal to all life

• redundancy: more than one codon code for each amino acids

Reading frame: the codons on the mRNA must be read in the correct groupings during translation to synthesize the correct proteins

Steps of Transcription

1) initiation 2) elongation 3) termination

Step 1: Initiation

• transcription begins when RNA polymerase molecules attach to a promoter region of DNA

• do not need a primer to attach

• promoter regions are upstream of the desired gene to transcribe

Eukaryotes:

• promoter region is called TATA box

• transcription factors help RNA polymerase bind

Prokaryotes:

• RNA polymerase can bind directly to promoter

Step 2: Elongation

• RNA polymerase opens the DNA and reads the triplet code of the template strand

• moves in the 3 prime to 5 prime direction

• the mRNA transcript elongates 5 prime to 3 prime

• RNA polymerase moves downstream

• only opens small sections of DNA at a time

• pairs complementary RNA nucleotides

• the growing mRNA strand peels away from the DNA template strand

• DNA double helix then reforms

• a single gene can be transcribed simultaneously by several RNA polymerase molecules

• helps increase the amount of mRNA synthesized

• increases protein production

Step 3: Termination

Prokaryotes

• transcription proceeds through a termination sequence

• causes a termination signal

• RNA polymerase detaches

• mRNA transcript is released and proceeds to translation

• mRNA does NOT need modifications

Eukaryotes

• RNA polymerase transcribes a sequence of DNA called the polyadenylation signal sequence

• codes for a polyadenylation signal (AAUAAA)

• releases the pre-mRNA from the DNA

• must undergo modifications Eukaryotes before translation

Pre-mRNA modifications

• there are three modifications that must occur to eukaryotic pre-mRNA before it is ready for translation

1) 5’ cap 2) poly-A tail 3) RNA splicing

• 5’ cap (GTP): the 5’ end of the pre-mRNA receives a modified guanine nucleotide “cap”

• poly-A tail: the 3’ end of the pre-mRNA recieves 50-250 adenine nucleotides

  • both the 5’ cap and the poly-A tail function to :

    • help the mature mRNA leave the nucleus

    • help protect the mRNA from degradation

    • help ribosomes attach to the 5’ end of the mRNA when it reaches the cytoplasm

• RNA Splicing: sections of the pre-mRNA, called introns, are removed and then exons are joined together

  • introns: intervening sequence, do not code for amino acids

  • exons: expressed sections, code for amino acids

• a single gene can code for more than one kind of polypeptide

  • known as alternative splicing

• once all modifications ahve occurred, the pre-mRNA is now considered mature mRNA and can leave the nucleus and proceed to the cytoplasm for translation at the ribosomes

Topic 4: Translation

Translation

• translation: the synthesis of a polypeptide using information from the mRNA

  • occurs at the ribosome

  • a nucleotide sequence becomes an amino acid sequence

Transfer RNA

• tRNA has an anticodon region which is complementary and antiparallel to mRNA

• tRNA carries the amino acid that mRNA codon codes for

• the enzyme aminoacyl-tRNA synthetase is responsible for attaching amino acids to tRNA

Ribosomes

• translation occurs at the ribosome

• ribosomes have two subunits: small and large

• prokaryotic and eukaryotic ribosomal subunits differ in size

• the large subunit has three sites: A, P, and E

  • A site: amino acids exons site

    • holds the next tRNA carrying an amino acid

  • P site: polypeptide site

    • holds the tRNA carrying the growing polypeptide chain

  • E site: exit site

Translation

1) initiation 2) elongation 3) termination

Step 1: Initiation

• translation begins when the small ribosomal subunits binds to the mRNA and a charged tRNA binds to the start codon, AUG, on the mRNA

• The tRNA carries methionine

• next, the large subunit binds

• the first tRNA carrying methionine will go to the P site, every other tRNA will go to the A site first

Ste 2: Elongation

• elongation starts when the next tRNA comes into the A site

• mRNA is moved through the ribosome and its codons are read

• each mRNA codon codes for a specific amino acids exons

• since all organisms use the same genetic code, it supports the idea of common ancestry

1) codon recognition: the appropriate anticodon of the next tRNA goes to the A site

2) peptide bond formation: peptide bonds are formed that transfer the polypeptide to the A site tRNA

3) translocation: the tRNA in the A site moves to the P site, the tRNA in the P site goes to the E site and the A site is open for the next tRNA

Step 3: Termination

• termination occurs when a stop codon in the mRNA reaches the A site of the ribosome

• stop codons do not code for amino acids

• the stop codon signals for a release factor

  • hydrolyzes the bond that holds the polypeptide to the P site

  • polypeptide releases

  • all translational units disassemble

Topics 5 and 6

Gene Expression

• prokaryotes and eukaryotes must be able to regulate which genes are expressed at any given time

• genes can be turned “on” or “off” based on evironmental and internal cues

  • on/off refers to whether or not transcription will take place

• allows for cell specialization

Bacterial Gene Expression

Operons: a group of gene that can be turned on or off

• operons have three parts

  • promoter: where RNA polymerase can attach

  • operator: the on/off switch

  • genes: code for related enzymes in pathway

• operons can be repressible or inducible

  • repressible (on to off): transcription is usually on, but can be repressed (stopped)

  • inducible (off to on): transcription is usually off, but can be induced (started)

Regulatory gene: produces a repressor protein that binds to the operator to block RNA polymerase from transcribing the gene

• always expressed, but a low levels

• binding of a repressor to an operator is reversible

Allosteric Regulation

Allosteric activator: substrate binds to allosteric sit and stabilizes the shape of the enzyme so that the active sites remain open

Allosteric inhibitor: substrate binds to allosteric site and stabilizes the enzyme shape so that the active sites are closed (inactive form)

Repressible Operons

• the trp operon in bacteria controls the synthesis of tryptophan

• since it is repressible, transcription is active

  • it can be switched off by a trp repressor

    • allosteric enzyme that is only active when tryptophan binds to it

• when too much tryptophan builds up in bacteria, tryptophan is mroe likely to bind to the repressor turning it active, which will then temporarily shut off transcription for tryptophan

Inducible Operons

• the lac operon controls synthesis of lactase, an enzyme that digests lactose

• since it is inducible, transcription is off

  • a lac repressor is bound to the operator (allosterically active)

• the inducer for the lac repressor is allolactose

  • when present it will bind to the lac repressor and turn the lac repressor off (allosterically inactive)

    • the genes can now be transcribed

Eukaryotic Gene Expression

• the phenotype of a cell or organism is determined by a combination of genes that are expressed and the levels that they are expressed

• differences between cell types is known as differential gene expression

Chromatin Structure:

• if DNA is tightly wound, it is less accessible for transcription

• histone acetylation adds an acetyl group to histones, which loosens the DNA which allows it to be easier to transcribe, activate

• DNA methylation adds methyl groups to DNA, which causes the chromatin to condense, deactivate

Epigenetic Inheritance:

• chromatin modifications do not alter the nucleotide sequence of the DNA, but they can be heritable to future generations

  • modifications can be reversed unlike mutations

Transcription initiation:

• once chromatin modifications allow the DNA to be more accessible, specific transcription factors bind to control elements

  • sections of non coding DNA that serve as binding sites

  • gene expression can be increased or decreased by binding of activators or repressors to control elements

  • translation can be activated or repressed by initiation factors

  • MicroRNAs and small interfering RNAs can bind to mRNA and degrade it or block translation

RNA processing:

• alternative splicing of pre-mRNA

Eukaryotic Development

• during embryonic development cell division and cell differentiation occcurs

  • cells become specialized in their structure and function

  • morphogenesis: the physical process that gives an organism its shape

• cytoplasmic determinants: substances in the maternal egg that influence cells

• induction: cell to cell signals that can cause a change in gene expression

  • both cytoplasmic determinants and induction influence pattern formation

    • a “body plan” for the organism

      • homeotic genes: map out the body structures

• as cells differentiate, apoptosis plays a critical role

  • apoptosis: programmed cell death

    • allows structures to take their form

Topic 7 and 8

Mutations

mutations: changes in the genetic material of a cell, which can alter phenotypes

• primary source of genetic variation

• normal function and production of cellular products is essential

  • any disruption can cause new phenotypes

• changes cna be alrge scale or small scale

  • large scale: chromosomal chagnes

  • small scale: nucleotide subsitutions, isnertions, or deletions

Small Scale Mutations

point mutations: change a single nucleotide pair of a gene

• subsitution: the replacement of one nucleotide and its partner with another pair of nucleotides

  • silent: change still codes for the same amino acid

  • missense: change results in a different amino acid

  • nonsense: change results in a stop codon

framshift mutation: when the reading frame of the genetic information is altered

• disastrous effects to resulting proteins

  • insertion: a nucleotide is inserted

  • deletion: a nucleotide is removed

Large Scale Mutations

• mutations that affect chromosomes

nondisjunction: when chromosomes do not separate properly in meiosis

• results in the incorrect number of chromosomes

translocation: a segment of one chromosomes moves to another

inversions: a segment is reversed

duplications: a segment is repeated

deletions: a segment is lost