AP Biology Unit 6: Gene Expression and Regulation

Structure of DNA and RNA

Structure of DNA and RNA

DNA contains the five-carbon sugar deoxyribose which has a hydrogen atom attached to the 2’ carbon, while RNA contains the five-carbon sugar ribose, which has a hydroxyl (-OH) group attached to the 2’ carbon. This makes DNA much more stable than RNA, explaining why it is the carrier of genetic info between generations, while RNA has more temporary functions; like in transcription, mRNA carries the genetic info from the nucleus to the ribosome. DNA is a double helix in which the two strands are antiparallel. One strand of the DNA is oriented with the 5’ phosphate group at the start of the strand and the opposite strand has the 3’ hydroxyl group at the start of the strand. A purine on one strand is always paired with a pyrimidine on the opposite strand, and since purines have a double-ringed structure and pyrimidines have a single-ringed structure, this keeps the width of the double helix consistent. RNA is typically single-stranded but can fold to form three-dimensional structures in rRNAs in the ribosome and in tRNAs.

DNA Replication

The purpose of it is to ensure the continuity of genetic info between generations. Each of the original two strands in the double helix serves as a template for a new strand. Since each new double helix is composed of one strand from the original piece of DNA and one newly synthesized strand, this is semiconservative. To start it, the enzyme helicase first unwinds the two DNA strands in an area called the origin of replication (ori site). As part of the double helix is unwound, other sections of the double helix become more tightly wound, resulting in supercoiling in those areas. Topoisomerase enzymes make temporary nicks in the sugar-phosphate backbone of DNA to relieve this supercoiling and then reseal these nicks. RNA polymerase then synthesizes an RNA primer using a few complementary RNA nucleotides. New DNA nucleotides can then be added to this RNA primer. DNA polymerase adds new nucleotides to the 3’ hydroxyl group at the end of this RNA primer. DNA polymerase adds new nucleotides in the 5’ to 3’ direction, always connecting the 5’ phosphate on the new nucleotide to the 3’ hydroxyl on the growing nucleotide strand. Because DNA polymerase can only add new nucleotides in the 5’ to 3’ direction, and because the two strands of DNA are antiparallel, DNA must proceed slightly differently on the two strands.

Leading Strand Replication

On one strand of the double helix, DNA polymerase reads the original strand in the 3’ to 5’ direction and can add new nucleotides continuously in the 5’ to 3’ direction.

Lagging Strand Replication

The other strand of the double helix is oriented in the 5’ to 3’ direction, making the replication process on this strand more complicated. On this strand, DNA polymerase must proceed in the opposite direction in order to read the strand in the 3’ to 5’ direction. Replication on this strand occurs discontinuously, producing short fragments called lagging strand fragments (Okazaki fragments), which are then joined together by the enzyme ligase.

Central Dogma

Describes the typical flow of genetic info in a cell: DNA is transcribed into mRNA; mRNA is then translated into proteins by ribosomes.

RNA Polymerase

The enzyme that transcribes a DNA sequence into RNA molecules. The function of an RNA molecule depends on its structure and sequence.

mRNA (messenger RNA)

Is single-stranded and carries info from DNA to the ribosome. It contains three base pair sequences called codons, which are complementary to the DNA base pair sequence. These codons will specify specific amino acids during translation.

tRNA (transfer RNA)

Folds into a three-dimensional structure that acts as an adapter molecule during translation. One end of this will bind to a specific amino acid, while the other end contains an anticodon, which will pair with the appropriate mRNA codon at the ribosome during translation.

rRNA (ribosomal RNA)

Folds into a three-dimensional structure. rRNA and proteins form ribosomes that perform translation. This three-dimensional thing acts as a ribozyme, catalyzing the reactions needed in translation.

Ribozyme

An RNA molecule that acts like a protein enzyme in catalyzing biochemical and metabolic reactions within a cell.

Promoter

To start transcription, the enzyme RNA polymerase must bind to one of these noncoding DNA sequences, which do not code for any amino acids and instead serve as a binding site for RNA polymerase upstream from the start of the coding region of a gene. They are highly conserved in living organisms.

TATA Box

Most eukaryotic promoters contain this region, so named because it is rich in thymine and adenine nucleotides.

Transcription Factors

These proteins help RNA polymerase bind to the promoter sequence and begin transcription.

Transcription

The process in which genetic info in a sequence of DNA nucleotides is copied into newly synthesized RNA molecules. During it, RNA polymerase adds new RNA nucleotides in the 5’ to 3’ direction. The strand of DNA being transcribed by RNA polymerase is called the template strand. The newly synthesized RNA must be antiparallel to the template DNA sequence. The template strand of DNA is also known as the minus strand, noncoding strand, or the antisense strand because the sequence of the mRNA strand that will be read by the ribosome during translation is not identical to the sequence of the DNA strand from which it was transcribed but instead is the opposite. The strand of the double helix that functions as the template strand can vary depending on the gene being transcribed. Since prokaryotes do not have a nucleus, in most of them the mRNA transcript formed in transcription is immediately accessible to ribosomes and can be translated without delay.

pre-mRNA

The initial mRNA transcript of eukaryotes, it needs to be modified before it can leave the nucleus of the cell and travel to the ribosomes for translation. Three modifications must occur: the removal of introns and the joining of exons, the addition of a GTP cap to the 5’ end of the RNA, and the addition of a poly-adenine tail to the 3’ end of the RNA.

Introns

Noncoding RNA sequences. While the do not code for amino acids, some may function in the regulation of gene expression.

Exons

A coding region of a gene that contains the info required to encode a protein.

Spliceosomes

Structures made of small nuclear RNAs (snRNAs) and small nuclear ribonucleoproteins (snRNPs) that remove the introns from the pre-mRNA and then splice together the exons, which can be joined in different combinations to generate multiple RNA transcripts from the same gene.

Alternative Splicing

A post-transcriptional mechanism that allows genes to act as exons and introns at the same time. It gives eukaryotes the ability to generate a greater variety of RNA transcripts from just one gene than what can be generated from one gene in prokaryotes.

GTP Cap

To protect the 5’ end of the pre-mRNA transcript from degradation before it can be translated, this is added. Nuclear pores recognize this and allow mRNAs with it to exit the nucleus. This also helps in the initiation of translation when the RNA reaches the ribosome.

Poly-A Tail

The enzyme poly-A polymerase adds a string of adenine nucleotides to the 3’ end of the pre-mRNA transcript. This helps prevent degradation of the transcript. mRNAs with these that are longer tend to have longer durations in the cytosol, which allows more copies of the protein (that the mRNA codes for) to be generated.

Mature mRNA

After the excision of the introns and splicing of the exons and the addition of the 5’ GTP cap and the 3’ poly-A tail, the transcript is referred to as this and is ready to be translated by the ribosome.

Translation

Occurs at the ribosomes in both prokaryotes and eukaryotes because they are found in the cytoplasm of both; as well as the rough ER of eukaryotes. In eukaryotes, cytoplasmic ribosomes usually do this to proteins that will stay inside the cell, while ribosomes on the rough ER usually do this to proteins that will be exported from the cell. Since prokaryotes do not have a nucleus, this can occur as the mRNA is being transcribed. Multiple ribosomes can be simultaneously translating a prokaryotic RNA, forming polyribosomes/polysomes. This process requires energy and involves three main steps in both eukaryotes and prokaryotes: initiation of translation, elongation of the polypeptide chain, and termination of translation.

Initiation

The genetic code in mRNA is read in three base pair units called codons. Translation is initiated when the rRNA in the ribosome pairs with the start codon (AUG). A tRNA with the complementary anticodon (in this case, UAC) brings the appropriate amino acid to the ribosome, and the anticodon on tRNA pairs with the codon on the mRNA. In this way, tRNA functions as an adapter molecule, linking the correct amino acids with the correct codon on the mRNA.

Elongation

After the first amino acid is placed in the ribosome by tRNA, the ribosome translocates (moves) to the next codon. A new tRNA with the appropriate anticodon and amino acid then pairs with this codon. The ribosome then catalyzes the formation of a peptide bond between the amino acids brought to the ribosome by the first two tRNAs, forming the beginning of the polypeptide chain. Once the peptide bond is formed between the amino acids, the first tRNA releases its amino acid (which is now linked to the second amino acid), and the first tRNA is released from the ribosome. This is repeated one codon at a time until a stop codon is reached.

Termination

Stop codons (nonsense codons) do not code for any amino acid. When the ribosome reaches a stop codon, proteins called release factors bind to the ribosome, causing it to disassemble and release the polypeptide chain, ending translation.

Flow of info from the nucleus to the cell membrane

In most organisms, genetic info starts with DNA, which provides the info for the transcription of mRNA. mRNA then provides the info for the sequence of amino acids in a protein. In eukaryotes, genetic info in DNA is transcribed into mRNA in the nucleus. Ribosomes on the rough ER use the info in mRNA to translate proteins. After this, a vesicle containing the protein will bud off from the rough ER and travel to the Golgi, where the proteins will be modified and packaged into vesicles for export from the cell. These vesicles will bud off from the Golgi and travel to the cell membrane. The vesicles then fuse with the cell membrane and release their protein contents from the cell.

Retroviruses

Viruses that contain RNA as their primary carrier of genetic info; they contain the enzyme reverse transcriptase.

Reverse Transcriptase

Makes a DNA copy of the RNA genome of the virus which is then inserted into the genome of the host cell that is infected by the virus. The host cell will then transcribe and translate the info in the viral DNA inserted into the host cell’s genome. They are less accurate than RNA polymerase, so retroviruses have a relatively higher mutation rate.

Phenotype

The observable characteristics in an individual resulting from the expression of genes. It is determined by the genes that are expressed and the levels at which those genes are expressed. Regulation of gene expression is important in determining this for an organism.

Regulatory Proteins

Proteins that can turn on or off genes by binding to specific nucleotide sequences.

Regulatory Sequences

The nucleotide sequences to which these regulatory proteins bind.

Regulation of Gene Expression in Prokaryotes

yes

Operon

What prokaryotes use to regulate gene expression. It is a cluster of genes with a common function under the control of a common promoter. They contain regulatory sequences, genes for regulatory proteins, and genes for structural proteins (which are responsible for the function of this).

Promoters

Noncoding regulatory sequences that serve as binding sites for RNA polymerase.

Operators

Noncoding regulatory sequences that serve as binding sites for repressor proteins (a type of regulatory protein).

Structural Genes

Coding sequences that contain the genetic code for the proteins required to perform the function of the operon.

Catabolism/Catabolic

The breakdown of organic molecules, producing useable forms of energy, such as ATP.

Inducible Operons

Usually have a catabolic function (digesting molecules) and are turned off unless the appropriate inducer molecule is present. The repressor protein in this binds to the operator sequence, blocking transcription of the operon by RNA polymerase. But, when an inducer molecule is present, the inducer binds to the repressor protein, changing its shape so that it can no longer bind to the operator sequence, allowing RNA polymerase to begin transcribing the operon.

Lac Operon

An example of an inducible operon, its function is to produce the proteins required to digest the sugar lactose. If no lactose is present, the lac repressor protein will bind to the operator, blocking transcription of the operon by RNA polymerase. Lactose serves as the inducer molecule for this. When lactose is present, it binds to the lac repressor protein, changing its shape so that it no longer can bind to the operator sequence, allowing RNA polymerase to transcribe the genes for the proteins that digest lactose. After all the lactose has been digested, the repressor can again bind to the operator sequence, shutting down the operon. This type of feedback mechanism allows the cell to manufacture the proteins needed to digest lactose only when they are needed, saving valuable resources in the cell. There are also ways to positively upregulate gene expression, shown through the interaction between cyclic AMP (cAMP) and the catabolite activator protein (CAP) in this. When glucose levels are low, cAMP levels in the cell increase. cAMP binds to the CAP, stimulating CAP to bind at a CAP binding site near the promoter. This increases the affinity of RNA polymerase for the promoter, stimulating transcription. So transcription of this is increased when glucose, another food source for the bacteria, is absent, allowing the cell to utilize the energy in lactose more efficiently.

Anabolism/Anabolic

The process by which the body utilizes the energy released by catabolism to synthesize complex molecules.

Repressible Operons

Usually have an anabolic function (synthesizing molecules) and are turned on unless the product of the operon is in abundance in the cell.

Trp Operon

An example of a repressible operon, its function is to produce the enzymes needed to synthesize the amino acid tryptophan. In this, the amino acid tryptophan functions as a corepressor. The trp repressor protein cannot bind to the operator sequence on its own; it must be bound to the amino acid tryptophan before it can bind to the operator. Therefore, if no tryptophan is present, the trp repressor will not be bound to the operator and RNA polymerase can transcribe the operon. When tryptophan is present, it will bind to the trp repressor, which will then bind to the operator, stopping transcription of the operon. This type of feedback mechanism allows the cell to make the enzymes needed to synthesize tryptophan only when the cell needs them, saving valuable resources in the cell.

Regulation of Gene Expression in Eukaryotes

no

Promoters

Sequences that serve as binding sites for RNA polymerase.

Regulatory Switches

Sequences to which activator proteins or repressor proteins may bind.

Enhancers

Regulatory switches to which activator proteins or transcription factors bind.

Silencers

Regulatory switches to which repressor proteins bind.

Repressors

Regulatory proteins that bind to regulatory switches and turn off or suppress gene expression.

Activators

Regulatory proteins that bind to regulatory switches and upregulate gene expression.

Transcription Factors

Regulatory proteins that help RNA polymerase bind to the promoter and start transcription.

Mediators

Regulatory proteins that serve as connectors between other regulatory proteins and allow regulatory proteins to communicate.

Epigenetic Changes

These can affect gene expression in eukaryotes. They can be reversible modifications to the nucleotides of the DNA sequence, such as methylation (adding a methyl group) of nucleotides. A methylated nucleotide is much less likely to be transcribed, so the cell can modify gene expression by changing the level of methylation of the nucleotides in various genes.

Histone Proteins

DNA is packaged into chromosomes around these. They can be epigenetically modified by adding acetyl groups (acetylation) to these. If these are acetylated, DNA will be more loosely wound around these, more accessible to RNA polymerase, and more likely to be expressed.

Euchromatin

DNA that is more loosely wound around the histone proteins, is more accessible to RNA polymerase, and usually results in more expression of the genes in this.

Heterochromatin

When DNA in chromosomes is tightly wound around the histone proteins, this is formed, which is less accessible to RNA polymerase and results in reduced gene expression.

Small Interfering RNA (siRNA) Molecules

Can affect gene expression; it is single-stranded and binds to complementary mRNA molecules, forming double-stranded RNA (dsRNA) molecules. Enzymes in the cell detect and destroy these dsRNA molecules, resulting in no translation of the targeted mRNA molecules and reduced gene expression.

Differential Gene Expression

The regulation of gene expression results in different genes being expressed in different cells, influencing the functions of cells and the resulting phenotype of the organism. Different tissue types express different genes, resulting in cell differentiation. The phenotype is not only determined by which genes are expressed but also by the levels at which the genes are expressed. The timing of the expression of different transcription factors during development is critical to the formation of specialized tissues and organs from a single-celled zygote. Errors in the timing of the expression of these genes can result in errors in the body plan or structures of an organism.

Aneuploidy

An incorrect number of chromosomes.

Mutations

Are changes in the genetic material of an organism. They may result in changes to the organism’s phenotype. Phenotype can change if this interferes with or changes the function of a protein. Those that change the amount of a protein produced can also change the phenotype of an organism. They provide genetic variation in populations which are acted upon by natural selection and can lead to the evolution of populations. Not all are harmful; the organism’s environment determines whether it is beneficial, harmful, or has no effect on the survival of the organism. If the environment changes, so does the benefit or harm this does to an organism. Some do not change the amino acid sequence of a protein at all because of the redundancy of the genetic code. They can be caused by environmental factors, like chemicals or radiation, or by random errors in DNA replication or DNA repair mechanisms. Mistakes in mitosis or meiosis can also lead to these. Failure of homologous chromosomes to separate during meiosis can lead to an aneuploidy, which in animals can be fatal or may cause sterility or other issues while in plants, aneuploidies that result in polyploids (extra set of chromosomes) can confer an advantage to the plant, making it more likely to survive.

Horizontal Transmission

Genetic mutations that are transmitted horizontally between members of the same generation: including transformation, transduction, conjugation, and transposition.

Transformation

The uptake of naked foreign DNA by a cell.

Transduction

The transmission of DNA from one organism to another by viruses; as the virus transfers the DNA, the DNA sequence may be recombined or otherwise changed, leading to new mutations and variations.

Conjugation

The transmission of DNA through cell-to-cell contact, usually through a connection called a pilus.

Transposition

The movement of DNA between chromosomes or within a chromosome.

Biotechnology

The tools of this are being used for many purposes: cancer therapies, improvement in agriculture yields, gene therapies for genetic disorders, de-extinction projects, and extinction projects.

Bacterial Transformation

Introduces foreign DNA into bacterial cells. The foreign DNA, usually a small, circular piece of DNA called a plasmid, may integrate into the host cell’s chromosome or remain separate from the host cell DNA in the cell’s cytoplasm.

Heat Shock

One method of transforming bacteria involves this, in which bacterial cells are mixed with foreign DNA and then quickly exposed to a cold-hot-cold temperature transition. This creates temporary microscopic pores through which the foreign DNA can enter some of the bacterial cells.

Plasmid DNA

This foreign piece of DNA needs a selectable marker so that cells that have incorporated this can be detected. The selectable marker is usually an antibiotic resistance gene that is not present in nontransformed bacterial cells. By growing the transformed bacteria on an agar plate that contains the antibiotic, one can select for the bacteria that absorbed and are now expressing the genes from this.

Restriction Endonucleases

Bacteria-produced enzymes that can slice between two DNA strands at areas called recognition sites.

DNA Ligases

Catalyzes the joining of adjacent polynucleotides; it is a DNA-joining enzyme.

Recombinant DNA

If the plasmid contains a gene from another organism, it is called this, meaning it is DNA that has been recombined from different source organisms. DNA can be cut at specific sequences using restriction endonucleases, and these pieces of DNA can be recombined and connected with DNA ligases.

Recombinant Plasmids

Some may contain the selectable marker as well as a gene that codes for a desired protein product. Bacteria that take in this would be capable of producing the product that is coded for by the gene on the plasmid. Many pharmaceutical products (such as insulin) are now produced in large amounts by bacteria that contain these.

Gel Electrophoresis

A technique that is used to separate DNA fragments by size and charge. DNA fragments can be created by treating a DNA sample with restriction enzymes, which cut DNA at specific base pair sequences. Due to the redundancy of the genetic code, even organisms with the same protein sequences will have slightly different DNA sequences. Treatment of DNA with restriction enzymes will cut these different DNA sequences at different locations and result in different fragment sizes. The backbone of the DNA double helix consists of five-carbon deoxyribose sugars and phosphate groups. The phosphate groups have a slight negative charge. DNA samples are loaded into wells at the top of the gel, and an electric current is applied to the gel. A positive cathode is attached to the bottom of the gel and the negative anode to the top of the gel. DNA molecules have an overall negative charge (due to their abundance of phosphate groups), causing them to migrate towards the bottom (positive end) of the gel. The gel itself is usually made of agarose and contains microscopic pores through which the DNA fragments migrate. Shorter fragments will be able to travel more quickly through these pores, leading to them being found at the bottom of the gel, farthest from the well into which the DNA sample was loaded; longer fragments will be closer to the top of the gel.

DNA Marker

The addition of this with fragments of known lengths runs alongside the other DNA samples, providing a ruler by which to estimate the size of each fragment produced from gel electrophoresis. Each organism’s pattern of fragments on the gel will be different and can be used to create a unique DNA fingerprint.

Polymerase Chain Reaction (PCR)

Is used to amplify specific DNA fragments. It can be used to create millions of copies of a specific fragment of DNA. It involves cycles of DNA replication using primers that are specific to the beginning and end of the fragment of the DNA sequence that is to be amplified. Each cycle of it doubles the number of copies of the desired DNA sequence. After just twenty cycles, over one million copies of the DNA sequence can be generated. It can also be used to rapidly sequence segments of DNA. Each cycle of it consists of three stages: denaturing the DNA, annealing of the primers, and extension of the primers.

2^n

The number of copies of the DNA sequence produced at the end of PCR is equal to this, where n is the number of cycles of PCR completed.

Denaturing the DNA

This separates the two strands of the double helix. It is usually done at a high temperature.

Annealing of the Primers

Once the DNA strands have been separated, the temperature is lowered slightly, and primers that are complementary to the desired DNA sequence are allowed to anneal (form hydrogen bonds with) the beginning and end of the fragment of the DNA sequence that is to be amplified.

Extension of the Primers

DNA polymerase adds new nucleotides to the primers, creating two copies of the desired DNA sequence. A heat-stable DNA polymerase is used for this step so that it will not be denatured during the rise in temperature with each cycle of PCR.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9

This system functions in nature as an adaptive immune system in bacteria. Bacteria can be infected by viruses called bacteriophages; and when one infects a bacterium, it cuts up the viral DNA and inserts those pieces between short palindromic repeats in the bacteria’s DNA. This allows the bacteria to store info on which viruses have previously infected its cell. When another virus infects the bacteria, the bacteria compare the DNA from the new virus to these stored DNA sequences. If it finds a match, it cuts the DNA of the virus with the Cas9 enzyme. CRISPR can be used to edit DNA sequences. The Cas9 enzyme uses a piece of guide RNA to find where to cut DNA. By using synthetic guide RNA pieces that correspond to the desired location of the cut to be made, one can direct the Cas9 enzyme to cut at a specific DNA sequence. If this cuts in the middle of a gene or one of its regulatory regions, this can create a knockout of the gene in which the gene is no longer functional. Observing the effects of this can help scientists understand the function of that gene. Sometimes instead of a knockout, a different DNA sequence at the site cut by the Cas9 enzyme is desired. Inserting multiple copies of a donor DNA sequence into the cell allows the cell’s own DNA repair mechanisms to use the donor DNA as a template. The cell can then replace the DNA sequence at the cut site with a copy of the donor sequence.