AP BIO Unit 6 Gene Expression and Regulation

Chargaff rule: Amount of adenosine equals amount of thymine and amount of cytosine equals amount of guanine

purines: double ring structure (A,G)

pyrimidines: single ring structure (C, U, T)

base pairs are held together by hydrogen bonds

• adenine and thymine have 2 hydrogen bonds

• cytosine and guanine have 3 hydrogen bonds

DNA is a double stranded heliz

• backbone: sugar phosphate

• DNA strands are antiparallel

• one strand runs 5’ - 3’ other strand runs in opposite 3’ - 5’ direction

• 5’ end: free phosphate group

• 3’ end: free hydroxyl group

DNA is the primary source of heritable information

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

  • RNA is the primary source of heritable information in some viruses

Eukaryotic Cells DNA

• DNA found in nucleus

• linear chromosomes

Prokaryotic Cells DNA

• DNA is in nucleoid region

• chromosomes are circular

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

Plasmids

• plasmids replicate independently from the chromosomal DNA

• primarily found in prokaryotes

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

• 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 can express the genes acquired

  • helps with survival of prokaryotes

RNA

• ribonucleic acid

• single stranded

• A=U C=G

DNA

• deoxyribonucleic acid

• double stranded

• A=T C=G

DNA replicaiton

• DNA replicates during the S phase of the cell cycle

• 3 models for DNA replication: conservative, semi-conservative, and dispersive

Conservative Model:

• the parental strands direct synthesis of an entirely new double stranded molecule

• the parental strands are fully “conserved”

Semi-conservative Model:

• the two parental strands each make a copy of itself

• after one round of replication, the two daughter molecules each have one parental and one new strand

• 4.

• *the correct model

Dispersive Model:

• the material in the two parental strands is dispersed randomly between the two daughter molecules

• after one round of replication, the daughter molecules contain a random mix of parental and new DNA

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 rebonding with itself, proteins called single strand binding proteins (SSBPs) bind to the DNA to keep it open

  • topoisomerase 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, to the 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 III (DNAP III) attaches to each primer on the parental strand and moves in the 3’ to 5’ direction

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

  • the DNAP III that follows helicase is the leading strand and only needs 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

  • Okazaki fragments: segments of the lagging strand

• 6. After DNAP III forms an okazaki fragment, 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’ end, there is no way to finish replication on the 5’ end of a lagging strand

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

  • how are the genes on DNA protected from this?

    • 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

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

  • includes two stages: transcription and translation

  • DNA → (undergoes transcription) → RNA (undergoes translation) → becomes protein

Transcription

• Transcription is the synthesis of RNA using information from DNA

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

• occurs in the nucleus

Translation

• Translation is the synthesis of a polypeptide using information from RNA

• occurs at the ribosome

• a nucleotide sequence becomes an amino acid sequence

Types of RNA

• 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

  • Base pairing: A=U and C=G

  • the mRNA nucleotide triplets are called codons

    • codons code for amino acids

• 64 different codon combinations

• 61 code for amino acids

• 3 are stop codons: UAA, UAG, and UGA

• universal to all life

• redundancy: more than one codon codes for each amino acid

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

Steps of Transcription

• Initiation

• Elongation

• Termination

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

Elongation

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

• moves in the 3’ to 5’ direction

• 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

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 before translation

Pre-mRNA modifications

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

  • 5’ cap

  • Poly-A tail

  • RNA splicing

• 1. 5’ cap (GTP): the 5’ end of the pre-mRNA receives a modified guanine nucleotide “cap”

• 2. Poly-A tail: the 3’ end of the pre-mRNA receives 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

• 3. 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

• Why does splicing occur?

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

    • known as alternative splicing

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

Translation

• Translation is the synthesis of a polypeptide using information from the mRNA

• occurs at the ribosome

• a nucleotide sequence becomes an amino acid sequence

• tRNA is a key player in translating mRNA to an amino acid sequence

Transfer RNA

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

• tRNA carries the amino acid that the mRNA codon codes for

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

  • when tRNA carries an amino acid it is “charged”

Ribosomes

• translation occurs at the ribosome

• ribosomes have 2 subunits: small and large

• prokaryotic and eukaryotic ribosomal subunits differ in size

  • prokaryotes: small subunit (30s) large subunit (40s)

  • eukaryotes: small subunit (40s) large subunit (60s)

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

• A site: amino acid 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

• Initiation

  • translation begins when the small ribosomal subunit 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

• 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 acid

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

• Elongation occurs in steps

  • Codon recognition: the appropriate anticodon of the next tRNA goes to the A site

  • Peptide bond formation: peptide bonds are formed that transfer they polypeptide to the A site tRNA

  • Translocation: the tRNA in the A site moves to the P site, the tRNA in the P site goes to the E site. The A site is open for the next tRNA

• 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

Protein folding

• as translation takes place, the growing polypeptide chain begins to coil and fold

• genes determine the primary structure

  • primary structure determines the final shape

• some polypeptides require chaperone proteins to fold correctly and some require modification before it can be functional in the cell

Retroviruses

• Retroviruses are an exception to the standard form of genetic information

• information flows from RNA to DNA

  • uses an enzyme known as reverse transcriptase

    • couples viral RNA to DNA

    • the viral DNA is integrated into the host’s genome, where it acts as a template for the production of RNA during replication

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 environmental 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 genes 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 at low levels

• binding of a repressor to an operator is repressible

Allosteric Regulation: Activator

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

Allosteric Regulation: inhibitor

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

Repressible Operons

• example: trp operon

• 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 more likely to bind to the repressor turning it active, which will then temporarily shut off transcription for tryptophan

Inducible Operons

• example: lac operon

  • 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

• eukaryotic gene expression is regulated at different stages

• chromatic structure:

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

  • how can it be modified?

    • histone acetylation: adds acetyle groups to histones which loosens the DNA

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

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

  • explains why one identical twin may inherit a disease while the other does not

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

RNA processing

• alternative splicing of pre-mRNA

Translation initiation

• translation can be activated or repressed by initiation factors

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

Eukaryotic Development

• during embryonic development cell division and cell differentiation occurs

  • cells become specialized in their structure and function

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

• How do cells differentiate during early development?

  • 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

        • homoeotic genes: map out the body structures

  • as cells differentiate, apoptosis plays a critical role

    • apoptosis: programmed cell death

      • allows structures to take their form

Mutations

• mutations are 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 can be small scale or large scale

    • small scale: nucleotide substitutions, insertions, or deletions

    • large scale: chromosomal changes

small scale mutations

• point mutations: change in a single nucleotide pair of a gene

• substitution: 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

• frameshift 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

  • reults in incorrect number of chromosomes

• translocation: a segment of one chromosome moves to another

• inversions: a segment is reversed

• duplications: a segement is repeated

• deletionss: a segment is lost

Natural selection

• any time mutations occur, they are subject to natural selection

• genetic changes can sometimes enhance the survival of an organism

Increasing Genetic Variation

• prokaryotes can exchange genetic material through horizontal gene transfer

• if there is a mutation that is beneficial to the survival and reproduction of that prokaryote, then it can also be transferred

  • transformation: uptaking of DNA from a nearby cells

  • transduction: viral transmission of genetic material

  • conjugation: cell to cell transfer of DNA

  • transposition: movement of DNA segments within and between DNA molecules

Gel electrophoresis: a technique used to separate DNA fragments by size

• DNA is loaded into wells on one end of a gel and an electric current is applied

  • DNA fragments are negatively charged so they move towards the positive electrode

PCR

• polymerase chain reaction

  • a method used in molecular biology to make several copies of a specific DNA segment

  • segments of DNA are amplified

  • results can be analyzed using gel electrophoresis

DNA Sequencing

• the process of determining the order of nucleotides in DNA