Genetics Final Exam

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Protein Encoding Genes

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Genes that encode the amino acid sequence for a polypeptide. AKA Structural Genes

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mRNA

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RNA transcribed from a protein-encoding gene

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Alkaptonuria

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Genetics disorder where a person accumulates abnormally high levels of homogentisic acid (also called alkapton), which is secreted in urine causing it to turn black when exposed to air.

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Inborn error of metabolism

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Rare genetic disorders in which the body cannot properly turn food into energy. The disorders are usually caused by defects in specific proteins (enzymes) that help break down (metabolize) parts of food.

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Sense Codons

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Sequence of 3 bases that specifies a particular amino acid.

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Start Codon

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Usually the first codon in a AA sequence. AUG = Methionie

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Anticodons

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3-nucleotide sequences (in tRNA molecules) that are complementary to codons in mRNA.

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Peptide Bond in Polypeptide

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Peptide bond between carboxyl group in the last AA of the polypeptide and the amino group of the next AA to be added.

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N-terminus

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Amino-terminus = The first AA in a polypeptide

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C-terminus

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Carboxyl-terminus = the last AA in a completed polypeptide (COO-)

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Chaperone proteins

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Proteins that bind to the polypeptides and facilitate their proper folding.

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Secondary Structure of Polypeptide

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Alpha helix and the Beta sheet

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Tertiary structure of Polypeptide

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Spontaneous (thermodynamically) favorable, avoid water, ionic interactions among charged AAs, H-Bonding amoung AA, and weak bonding (van der Waals interactions)

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aminoacyl-tRNA sythetases

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Catalyze the attachment of AAs to tRNA molecules (uses ATP). ATP is hydrolyzed and AMP becomes attached to AA; pyrophosphate is released. Now tRNA is charged.

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isoacceptor tRNAs

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tRNAs that can recognize the same codon but differ at the wobble base

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Purpose of nucleolus (Euk.)

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Assembly of the rRNAs and ribosomal proteins to make the 40S and 60S subunits. Then exported to cytosol where they becomes the 80S ribosome during translation.

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Shine-Dalgarno sequence

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9-nucleotide sequence in the mRNA that facilitates the H-bonding of the mRNA to the 30S subunit (in bacteria). IF1 and IF3 bind to 30S subunit. Later IF2 (with GTP) promotes binding of the initiator tRNA

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How are the eukaryotic mRNAs recognized by the ribosome?

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No S-D sequence. The mRNA is recognized by eIF4, which is a multiprotein complex that recognizes the 7-methylguanosine cap at the 5' end of the mRNA. eIF4 then facilitates binding of the 5' end of the mRNA to the 40S subunit.

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eLF5

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Causes the release of the other initiation factors (eIF1, 3, and 6), which enables the 60S subunit to associate with the 40S subunit.

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eIF2

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Promotes the binding of the initiator tRNA to the small ribosomal subunit

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16S rRNA

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Subunit that can detect when an incorrect tRNA is bound at the A site and will prevent elongation until the mispaired tRNA is released from the A site.

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EF-G and eEF2

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Elongation factors required for translocation.

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RF1, RF2 and eRF1

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Release factors the recognize a stop codon and trigger the cleavage of the polypeptide from the tRNA. (RF3 and eRF3 are GTPases that are also involved in termination). Release factors are molecular mimics because their structures are similar to a tRNAs.

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Gene regulation

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Phenomenon in which the level of gene expression can vary under different conditions. Metabolism; response to environment; cell division.

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Constitutive genes

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Unregulated genes

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Repressor protein

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A regulatory protein that binds to the DNA and inhibits transcription. (Negative Control)

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Activator protein

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A regulatory protein that increases the rate of transcription. (Positive Control)

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Inducer

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Small effector molecule that cause transcription to increase (inducible genes i.e. lac operon)

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Corepressor

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a small molecule that binds to a repressor protein, thereby causing the protein to bind to the DNA. (Repressible genes)

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Inhibitor

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Binds to an activator protein and prevents it from binding to the DNA (Repressible genes)

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Enzyme adaptation

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Observation that a particular enzyme appears within a living cell only after the cell has been exposed to the substrate for that enzyme.

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Operon

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A group of two of more genes under the transcriptional control of a single promoter.

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polycistronic mRNA

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an RNA that contains the sequences of two or more genes.

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Promoter

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Signals the beginning of transcription

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Terminator

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Specifies the end of transcription.

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lacZ

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encodes the gene Beta-galactosidase and enzyme that cleaves lactose into galactose and glucose. Also converts a small percentage of lactose into allolactose (side reaction).

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lacY

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encode lactose permease, a membrane protein required for the active transport of lactose into the cytoplasm

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lacA

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encodes galactoside transacetylase, an enzyme that covalently modifies lactose and lactose analogs.

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CAP site

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a DNA sequence recognized by an activator protein called the catabolite activator protein (CAP).

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Operator site

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Operator is a sequence of bases that provides a binding site for a repressor protein (lac repressor)

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IacI

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gene that encodes the lac repressor, a protein that regulates the lac operon by binding to the operator site and repressing transcription. Homotetramer; a protein made of four identical subunits.

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Allolactose

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The inducer; when 4 molecules bind to the repressor a conformational change occurs that prevents the repressor from binding to the operator. (Operon has now been induced)

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Allosteric regulation

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The action of a small effector molecule binding to the allosteric sites of a repressor.

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lacI- mutation

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Constitutive expression of the lac operon, which means it is expressed in the presence and absence of lactose.

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lacI^s mutation

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The lac operon cannot be induced even in the presence of lactose. Super-repressor mutation. Lac repressor is unable to bind to allolactose, it will remain bound to the lac operon and induction cannot occur.

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Merozygote

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Partially diploid; a strain of bacteria containing F' factor genes. Genes are not physically adjacent to each other. However, a normal lacI gene can produce repressor proteins that can diffuse within the cell and eventually bind to the operator site of the lac operon located on the chromosome and also to the operator site on an F' factor

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trans-effect

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a form of genetic regulation that can occur even though two DNA segments are not physically adjacent. (The action of the lac repressor on the lac operon)

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trans-acting factor

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A regulatory protein, such as the lac repressor. Mutation in a trans-acting factor can be complemented by the induction of a second gene with a normal function.

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cis-effect

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cis-acting element = a DNA segment that must be adjacent to the gene(s) that it regulates. Mediated by DNA sequences that are binding sites for regulatory proteins (lac operator site). Mutation in a cis-acting element is not affected by the introduction of a normal cis-acting element into the cell.

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Catabolite repression (somewhat imprecise term)

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A form of transcriptional regulation influenced by the presence of a catabolite (a substance broken down inside the cell--like glucose)

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Diauxic growth

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The sequential use of two sugars

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cyclic-AMP (cAMP)

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a small effector molecule that is produced from ATP via an enzyme known as adenylyl cyclase. When glucose is transported into the cell it stimulates a signaling pathway that cause the intracellular concentration of cAMP to decrease because the pathway inhibits adenylyl cyclase.

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CAP

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Catabolite activator protein---Mediates the effect of cAMP on the lac operon. Composed of two subunits; each of which binds one molecules of cAMP.

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Lactose (present only) allolactose and cAMP levels are high

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1. Allolactose binds to the lac repressor and prevents it from binding to the DNA.

2. cAMP binds to the CAP

3. CAP binds to the CAP site and stimulates RNA polymerase to begin transcription.

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High cAMP but no lactose or glucose

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The binding of the lac repressor inhibits transcription even though CAP is bound to the DNA (no allolactose). Therefore, transcription rate is very low.

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Glucose & Lactose present

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Presence of glucose decreases cAMP levels so the cAMP is released fro CAP, which prevents CAP from binding to the CAP site. Because CAP is not bound to the CAP site, the transcription of the lac operon is low in the presence of both sugars.

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Effect of Glucose (Metabolically)

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Catabolite repression = puzzling. Because this regulation involves and inducer (cAMP) and an activator protein (CAP), not a repressor. Term was coined BEFORE we knew the entire process; primary observation was that glucose (a catabolite) inhibited (repressed) lactose metabolism.

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Three Operator Sites for lac repressor

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O1 operator site slightly downstream from the promotor. O2 is located farther downstream in the lacZ coding sequences. O3 is located slightly upstream from the promotor. The repressor must bind to two operator sites to repress the lac operon. O1 and O2 or to O1 and O3. But not O2 and O3. * A loop in the DNA brings the operator sites closer together, thereby facilitating the binding of the repressor protein.

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Attenuation

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Transcription begins, but is stopped prematurely, or "attenuated" before most of the trp operon is transcribed

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trpL

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Part of the trp operon and helps mediate attenuation.

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trpR

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Is not part of the trp operon and has its own promoter. Encodes the trp repressor protein.

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Low Trp levels

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The trp repressor cannot bind to the operator sites. RNA polymerase transcribes the trp operon.

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High Trp levels

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Tryptophan acts as a corepressor that binds to the trp repressor protein that allows it bind the operator site. Inhibits RNA polymerase.

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Posttranslational regulation

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refers to the functional control of the proteins that are already present in the cell rather than regulation of transcription or translation. (Can be relatively fast)

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Translational regulatory protein

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recognizes sequences within the mRNA. Typically act to inhibit translation so they're known as translational repressors.

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antisense RNA

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an RNA strand that is complementary to a strand of mRNA and therefore blocks its translation.

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Posttranslational covalent modification

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Can be irreversible to produce a functional protein. Or reversible like phosphorylation (-PO4), acetylation (-COCH3), and methylation (-CH3).

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Riboswitches

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regulation in which a RNA molecules can exist in two different secondary conformations. 3-5% of all bacterial genes may be regulated by riboswitches.

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Riboswitches (Transcription)

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The 5' region of the an mRNA may exist in one conformation that forms a rho-independent terminator, which causes attenuation of transcription. The other conformation does not form a terminator and is completely transcribed.

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Riboswitches (Translation)

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The 5' region of an mRNA ma exist in one conformation in which the Shine-Dalgarno sequence cannot be recognized by the ribosome, whereas the other conformation has an accessible S-D and allows translation.

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Riboswitches (RNA stability)

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One mRNA conformation may be stable, whereas the other acts as a ribozyme that causes self-degradation.

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Riboswitches (Splicing)

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In Euks, one pre-mRNA conformation may be spliced in one way, whereas another conformation is spliced a different way.

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Thiamin pyrophosphate (TPP)

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An essential coenzyme for the functioning of a variety of enzymes (citric acid cycle). Low level of TPP, the secondary structure has a stem-loop called an antiterminator which prevents the formation of the terminator stem-loop. (Gram-Positive bacteria) -- Riboswitch in transcription

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TPP Low in Gram-Negative Bacteria

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The 5' end of the mRNA folds into a structure that contains a stem-loop called the Shine-Dalgarno antisequestor (Riboswitch in translation)

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Recombinant DNA technology

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the use of in vitro molecular techniques that manipulate fragments of DNA to produce new arrangements.

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Gene Cloning

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The process of making many copies of a gene.

1) Isolate DNA (into a vector)

2) Make plasmid

3) Introduce to (competent) host cells

4) Transformation (DNA Ligase)

5) Incubate and plate on growth media

6) Selectable mark (i.e., antibiotic resistance) to select for recombinant colonies

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Uses of Gene Cloning

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1) DNA Sequencing: identifying gene sequences such as promotors, regulatory sequence, and coding sequences. ID of cancerous alleles and inherited human diseases

2) Site-directed mutagenesis

3) Gene probes

4) Expression of cloned genes (Research, Biotechnology, Clinical trials)

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Gene probes

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Labeled DNA strands from a cloned gene used to identify similar or identical gene or RNA. Methods include Northern & Southern blotting. Probes can also be used to localize genes within intact chromosomes.

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vector

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a small DNA molecule that can replicate independently of host cell chromosomal DNA and produce many identical copies of an inserted gene. (Acts as a carrier of the DNA segment that is to be cloned)

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host cell

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a cell that harbors a vector

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plasmids

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small circular pieces of DNA; found naturally in many strains of bacteria and occassionally in euk cells

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R factors

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naturally occurring plasmids carrying genes that confer resistance to antibiotics or other toxic substances

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origin of replication (gene cloning)

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DNA sequence in plasmids that is recognized by the replication enzymes of the host cell and allow it to be replicated. Determines whether or not the vector can be replicated in a particular type of host cells. Also determines copy number of a plasmid

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Selectable marker

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Often antibiotic resistance; a gene that selects for the growth of the host cells by expression.

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amp^R

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A gene that encodes an enzyme beta-lactamase: an enzyme that degrades ampicillin

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Viral vector replication

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Viruses infect living cells and propagate themselves by taking control of the host cell's metabolic machinery. Analyze viral plaques instead of bacterial colonies

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Other types of vectors

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cosmids, bacterial artificial chromosomes (BACs), and yeast artifical chromosomes (YACs) -- Used to clone large pieces of DNA

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Restriction endonucleases (enzymes)

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Used in cloning experiment to bind to a specific bas sequence and then cleave the DNA backbone at two defined locations, one in each strand. Key step in the insertion of chromosomal DNA into a plasmid or viral vector. Naturally made by bacterial cells to protect against invasion by foreign DNA (particularly bacteriophages) Used to digest into "sticky ends"

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Hydrogen bonding between the sticky end in cloning process

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Temporary interaction; not stable because it involves only a few hydrogen bonds. Cue DNA ligase.

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Restriction enzymes usually recognize sequences...

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Two-fold rotational symmetry (NOT palindromic)

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Recombinant vector

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vector containing a piece of chromosomal DNA

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Reverse Transcriptase

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Used to make cDNA. Uses RNA as a template to make a complementary strand of DNA. mRNAs are mixed with primers composed of a string of thymine-containing nucleotides

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Oligonucleotide

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Poly-dT primer; complementary to the 3' end of the mRNAs. Reverse transcriptase and dNTPs.

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Ways to make the other cDNA strand

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Use RNaseH, which partially digests the RNA, generating short RNAs that are used as primers by DNA polymerase to make a second DNA strand that is complementary to the strand made by reverse transcriptase

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cDNA

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DNA that is made form RNA as a starting material (double or single-stranded). Lacks introns (often found in Euk genes). Much simpler to insert into vectors.

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DNA library

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Collection of recombinant vectors, with each vector containing a particular fragment of chromosomal DNA. When starting material is chromosomal DNA = genomic library

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cDNA library

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Collection containing recombinant vectors with cDNA inserts. To insert the cDNAs into vectors, short oligonucleotides called linkers are attached to the cDNAs via DNA ligase. After attachment the cDNAs and vectors are cut w/ restriction enzymes and then ligated to each other.

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Differences Between Western and Northern/Southern Blotting

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Use of an antibody as a probe, rather than a labeled DNA strand. Antibodies bind to sites known as epitopes.

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Epitopes

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has a three-dimensional structure that is recognized by an antibody. In the case of proteins, an antigen is a short sequence of amino acids.

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Antigen

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refers to any molecule that is recognized by an antibody. An antigen contains one or more epitopes.

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Primary antibody

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AA sequence is unique for each protein, any given antibody specifically recognizes a particular protein. (in western blotting)

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Secondary antibody

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(In western blotting) After the primary antibody has been given sufficient time to recognize the protein of interest, any unbound primary is washed away and a secondary is added.

- an antibody that specifically recognizes and binds to a region in the primary antibody; may be labeled or conjugated to an enzyme (common enzymes akaline phosphatase)

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Gel mobility shift assay

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technique for detecting interaction between RNA-binding proteins and mRNAs and between transcription factors and DNA regulatory elements. In the case of DNA binding proteins; the binding of a protein fragment retards the fragment's ability to move through a polyacrylamide or agarose gel; because protein-DNA complex has a higher mass. Visualized by staining with dye such as EtBr or fluorescent molecule. *must be carried out under non-denaturing conditions*

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DNase I footprinting (Also works with euk transcription factors)

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identify one or more regions of DNA that interact with a DNA-binding protein. Compared to Gel Mobility Shift Assay; provides more detailed information about the interactions between a protein and DNA. (However, more complicated technique) DNase I cleaves covalent bonds in the backbone and used a reagent for determining if a DNA region has a bond protein (cannot cleave region where protein is bond).

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Rational behind DNA footprinting?

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Accessibility; if a protein is bound to the DNA it will shield the segment of the DNA it is bound to therefore will not allow DNase I to cut it into smaller fragments leaving a gap in the bps.

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Probe

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Can be a DNA probe that is complementary to the gene you want to clone or it can be and antibody that recognizes the protein that is encoded by the gene

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Steps in GENE Cloning

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1) Obtain desired chromosomal DNA from cell (tissue) sample

2) Obtain a vector

3) Digest the vector and chromosomal DNA w/ a restriction enzyme

4) Mix them together to allow the sticky ends to H-bond

5) Add DNA ligase to promote covalent bonding (hopefully create a recombinant vector)

6) Introduce to living cell

7) Vector will replicate and the cells will divide to produce colonies of cell

8) ID colonies with correct recombinant using a probe

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Important Features of Cloning Vectors

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Ability to replicate when introduced into a living cell. Origin of replication that determines the host cells specificity of a vector. Modern vectors have convenient restriction sites where you can insert DNA fragments. Selectable markers (confer some selectable advantage for the host cell that carries them)

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Possible uses of site-directed mutations

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Study the core promoter of a gene; study regulatory elements; identify splice sites; study the important of particular AAs with regard to protein function

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After four cycles of PCR, which types of PCR product predominates? Why?

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The segments flanked by promoters; because these segments provide templates to make products only like themselves.

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The Wobble Rules

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1) Wobble position occurs at the 3rd base in the mRNA codon & corresponding base in the anticodon

2) The structures in bases found in tRNA may be modified

Third Base of mRNA Codon & Base in anticodon can be: A --- U, I, xm^5Um, Um, xm^5 xo^5U, k^2C

U --- A, G, U, I, xo^5U

G --- C, A, U, xo^5U

C --- G, A, I

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lac Operon Regulation (No sugar present)

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The operon is turned off due to the dominating effect of the lac repressor protein

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lac Operon Regulation (Lactose)

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The operon is maximally turned on. The repressor protein is removed from the operator site, and the CAMP-CAP complex is bound to the CAP site

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lac Operon Regulation (Glucose)

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The operon is turned off due to the dominating effect of the lac repressor protein.

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lac Operon Regulation (Lactose & Glucose)

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The expression of the lac operon is greatly decreased. The lac repressor is removed from the operator site, and the catabolite activator protein is not bound to the CAP site. The absence CAP at the CAP site makes it difficult for RNA polymerase to being transcription. However, a little more transcription occurs under these conditions than in the absence of lactose, when the repressor is bound.

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Peptidyl transferase

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Is part of the ribosome itself

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I^s is....

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super repressor (inducer)

I^s acts in trans and represses both wild-type operators. Since the allolactose binding site on lacI^s is defective, it cannot be induced.

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Restriction Endonuclease Class II site example

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5' GATC 3' / 3' CTAG 5'

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Which of the following sequences would not be in a genomic library?

A Promotor

Enhancer/Silencer

Telomere

Introns

Poly A Tail

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Poly A Tail (Because it is not processed RNA)

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Mutant tRNAs may act as nonsense & missense suppressors. How do they work?

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For example: 1st mutation encode stop codon. 2nd mutation in a tRNA gene can alter the anticodon region of a tRNA so that the anticodon recognizes a stop codon but inserts an amino acid at this site.

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tRNA supressor

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is a second-site mutation that suppresses the phenotypic effects of a first mutation.

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Intergenic supressor mutations

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in tRNA genes act as nonsense or missense suppresor within the same gene as the first mutation

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Missense supressor

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a mutation in a tRNA gene that changes the anticodon so that it puts it in the wrong AA at a codon that is not a stop codon. Can be produced by mutations in the anticodon region of tRNAs so the tRNA recognizes an incorrect codon (or) can be produced by mutations in aminoacyl-tRNA sythetases that cause them to attach the incorrect amino acid to a tRNA