Genetics Test 4 Chapters 14-16

codon

a three-nitrogenous bases (nucleotides) sequence of DNA (a triplet) that codes for an amino acid

Start codon

AUG, methionine

Stop codons

UAA, UAG, UGA, which don’t code for any amino acids

Reading frame

a linear sequence of codons in a nucleic acid defined by a start codon and ending with a stop codon

Characteristics of the Genetic Code: Unambiguous

each of the 61 triplets code for only one of the 20 amino acids

Characteristics of the Genetic Code: Degenerate

most amino acids are encoded by more than one codon

Characteristics of the Genetic Code: Universal

Most living organisms use the same code (but exceptions exist)

Characteristics of the Genetic Code: Commaless

there are no breaks between the codons in a reading frame (all the bases of the translated sequences are part of codons)

Characteristics of the Genetic Code: Non-overlapping

the triplets in a reading frame are in a tandem sequence and do not overlap.

antisense

DNA template strand

substitution mutation

occurs when a base is replaced with a different base in DNA resulting in a permanent single-codon change in a protein coding-gene

silent mutation

a type of substitution mutation. The resulting new codon codes for the same amino acid as the original codon

Missense Mutation

a type of substitution mutation. The new codon codes for a different amino acid

Nonsense mutation

a type of substitution mutation. The new codon is a stop codon, resulting in a shorter phenotype

mutations may be

spontaneous, or may be caused by errors in DNA replication

mutations occur in

the DNA, and are copied to RNAs if they occur within genes

Insertion Mutation

due to errors in DNA replication, where a nucleotide is added. All the codons from the site are changed, resulting in a frameshift.

Basic requirements of translation

• mRNA

• Charged transfer RNAs (tRNA)

• Ribosome

• initiation factors, elongation factors

• Energy sources

• No primers

Transfer RNA (tRNA)

a single RNA with intramolecular base pairings. An amino acid is attached to the 3’ end of this RNA.

the anticodon loop base-pairs with

a codon in an mRNA

Inosine (I)

a modified adenine

post-transcriptional modification

editing of bases in tRNAs resulting in unusual bases

The wobble hypothesis

a limited number of tRNA anticodons can recognize multiple mRNA codons by allowing non-standard base pairing at the third codon position. This allows translation to occur without the need for the cell to synthesize all 61 tRNAs

Anticodon-Codon Base Pairing Rules

Anti-parallel base pairing of the codon and anticodon. The third base pair, the wobble base pair, can pair with other bases they usually wouldn’t.

charging

the carboxyl group of the amino acid is covalently attached to the 3’ end of the tRNA. It is catalyzed by twenty different Aminoacyl tRNA synthases

energy is spent in the process of charging. It eventually makes

a peptide bond in the ribosome

Steps of charging of a tRNA

1. Amino Acid activation

2. Charging

Amino Acid activation

the amino acid is converted to an aminoacyl adenylic (AA-AMP), which is the energy consuming step in the process of making a new peptide bond.

Charging tRNA

the aminoacyladenylic acid loses the AMP and the carboxyl group of the amino acid is attached to the 3’ end of a tRNA. The result is an aminoacyl tRNA (a charged tRNA).

ribosome

large particle of rRNA and proteins where translation occurs. Two subunits, small and large.

Svedberg Unit (S)

not a measure of molecular weight, but a measure of the rate at which particles sediment in a centrifugal field. Depends on weight, shape, and size, and can be used for measuring large molecules or large cell components such as ribosomes and organelles

Svedburg Values: Small subuint

30S (prokaryotic ribosomes)

Svedburg Values: Large Subunit

50S (prokaryotic ribosomes)

Svedburg Values: Complete ribosome

70S (prokaryotic ribosomes)

Sites in the ribosome

• Peptydyl (P)

• Aminoacyl (A)

• Exit (E)

Steps in the initiation of translation

1. the mRNA binds to the small ribosomal subunit with the AUG codon positioned on the P site

2. f-Met-tRNA (in prokaryotes) binds to the AUG codon

3. The large ribosomal subunit joins the complex. This process requires GTP for a source of energy plus a series of initiation factors (IF proteins)

Formyl-methionine

A starting protein in bacteria and eukaryotic organelles

In mRNA transcription

the small ribosome attaches first, as the IF-3 restricts the large subunit initially. After the tRNA and IF-1 join, then the large subunit can join.

EF-Tu

an elongation factor and GTP facilitate the binding of the second tRNA to the second codon at the A site

After EF-Tu connects,

the amino acid on the first tRNA is transferred and forms a peptide bond with the amino acid on the second tRNA

dipeptide form

when the amino acid on the first tRNA is transferred to the second tRNA, and forms a peptide bond

Elongation of the polypeptide chain: Steps

• EF-Tu and GTP facilitate the binding of the second tRNA to the second codon at the A site

• The amino acid on the first tRNA is transferred and forms a peptide bond with an amino acid on the second tRNA (dipeptide form)

• First tRNA moves to E site (exit)

• mRNA is shifted to place the second codon in the P site and brings the codon into the A site

• tRNA carrying the third amino acid binds the third codon on the A site

• forms tripeptide attached to second tRNA and moves to E site

Elongation of the polypeptide chain: Simplified

tRNA attaches to start codon (AUG) in the P zone. A second tRNA attaches to the A zone, forming a dipeptide. The ribosome moves forwards, the first tRNA goes to the E zone, and exits.

termination of translation

A STOP codon moves to the A site of the ribosome, which doesn’t code for any proteins. Release factor 1 binds to the stop codon, which disconnects the peptide chain. Release factor 3 releases the ribosome and dissociates the entire process.

The Mechanisms of gene regulation

determine where when and how much a gene is expressed

Every gene is an

RNA-coding region (a transcribed region) of DNA. Nearby regulatory region(s) are not transcribed

Transcription factors

DNA binding proteins that recognize specific sequences within the regulatory region(s) near the gene to either activate or repress transcription

operon

a cluster of structural genes with related functions under the control of a common regulatory system that can respond to changes in environmental conditions. Common in prokaryotes but rare in eukaryotes.

Examples of structural genes under operon control

lacZ, lacY, lacA

the lac operon

Genes only expressed if lactose is available, but also, only if the cell needs to use lactose for energy. The cell prefers to use glucose

glucose

the preferred source of energy in the cell

lacZ gene product

Beta-galactosidase. an enzyme that breaks down lactose (a disaccharide) into galactose and glucose. can also isomerize lactose into allolactose

lacY gene product

permease. A membrane transporter for lactose, facilitates the entry of lactose into the bacterial cell

lacA gene product

transacetylase. Not involved in lactose metabolism, involved in the removal of by-products of lactose digestion from the cell

Control regions for the Lac operon

lacP (promoter) and lacO (operator)

lacP (promoter or Plac)

binding site for RNA polymerase

lacO (lac operator)

binding site for the regulator protein; it overlaps lacP

Regulator locus in lac operon

lacI and PI

lacI

the gene that codes for the regulator protein

PI

the promoter for the lacI gene

In the absence of lactose

the regulator protein, a repressor binds to the operator, blocking RNA polymerase from binding to the promoter

State of the lac operon in the absence of lactose

the regulator protein a repressor binds to the operator, blocking RNA polymerase from binding to the promoter

Leaky

very small amounts of Beta-galactosidase and permease are still made, even if glucose is available to the cell

allolactose

the operon inducer. Binds to the repressor protein and inactivates it, which activates the expression of the structural genes.

If glucose is available to a cell

the lac structural genes are off, even if lactose is present. The binding of RNA polymerase to the promoter occurs only if glucose is absent.

Effect of glucose levels on CAP activity

The activity of the enzyme adenylate cyclase, which catalyzes the hydrolysis of ATP into cAMP + PP is induced when glucose is absent. Cytoplasmic levels of cAMP increases, which activates CAP

catabolite activated protein

CAP. Activated by cAMP, and binds to the site next to the lac promoter and facilitates the binding of RNA polymerase to it. Necessary for the stable binding of RNA polymerase to the lac promoter, only active when glucose is absent

cAMP

is the catabolite that binds to the catabolite activated protein (CAP) to activate it.

In the presence of glucose

cAMP levels decrease, and CAP remains inactive. RNA polymerase does not bind to the promoter efficiently, and the operon is OFF

The products of the five structural genes of the trp operon

are enzymes involved in the biosynthesis of the amino acid tryptophan (Trp)

If Trp is absent

the structural genes are expressed. The repressor protein remains inactive, and the RNA polymerase binds to the promoter and initiates the transcription of the structural genes

If Trp is present

the structural genes are turned off by Trp itself. Trp binds to the repressor protein and activates it. The binding of the Trp-activated repressor to the operator prevents the binding of RNA polymerase to the promoter

Electrophoretic Mobility Shift Assay (EMSA)

a method used for detecting specific DNA-protein interactions, such as the specific binding of transcription factors and other regulatory proteins to regulatory regions of the chromosome

example of EMSA (mixed in snap cap/test tube)

Fragment of DNA containing trpO, E. coli cell extract which contains all cellular proteins, trypyophan (Trp), specific antibodies against trp repressor and other cellular proteins, such as the lac repressor

Electrophoretic mobility shifts

occur due to the different sizes of the complexes that form based on which antibodies attach to the DNA

RNA polymerase 1

the larger rRNA genes (5.8s, 18s, and 28s)

RNA polymerase 2

all the protein-coding genes

RNA polymerase 3

the small rRNA (5s) gene and all the tRNA genes

Heterochromatin

transcriptionally inert due to the tight association of DNA with histones, which prevents RNA polymerase from binding to gene promoters. The ability of the cell to alter the association of DNA with histones is essential to allow gene regulatory proteins and RNA polymerases to bind to DNA

Histone acetylation

weakens the interaction between basic histones and the acidic DNA molecule, causing chromatin decondensation. HATs and HDACs do not bind to DNA but regulate histone acetylation

histone acetyl transferases (HATs)

recruited by transcription activators. Causes chromatin decondensation. Act as coactivators, but they do not bind to DNA

Histone deacetylases (HDACs)

recruited by transcription repressors, causing chromatin condensation. Act as corepressors, but they do not bind to DNA

Flowering Locus C (FLC)

codes for a transcription factor that represses flowering but is only expressed if the histones on the locus are acetylated. Represses genes that promotes flowering in young plants.

Flowering locus D (FLD)

A gene product. A histone deacetylase that specifically inactivates the FLC locus to allow flowering

transcription factor

any transcription regulator protein that binds to a specific DNA sequence

cis-acting elements

DNA sequences that are necessary for the control of transcription (The regulatory regions of the chromosome) Promoters, enhancers, and silencers.

trans-acting factors

proteins that are necessary for the control of transcription. The transcription factors that bind to the cis-acting elements

Promoters

The binding site for the RNA polymerase 2 transcription initiation complex. A key component of the promoters of protein-coding genes in eukaryotes is a TATAAA sequence within them known as the TATA box

Enhancers

the binding sites for transcriptional activator proteins

silencers

the binding sites for transcriptional repressor proteins

enhancers

required for stimulated transcription. Their DNA sequences vary widely and are recognized by a large variety of transcription activators. Work at a distance, inverted, or on top of a promoter.

The activation of a gene in any particular cell

depends on whether the cell has the right activators to bind to the gene enhancers

Promoter alone

only the basal transcription factors may bind to DNA, and only the basal transcription apparatus may form. Only very low or undetectable transcription can occur

With the right enhancers

transcriptional activators bind to enhancers. Stimulated transcription (biologically significant).

While enhancers can be moved around

promoters cannot be moved

insulators

Also known as boundary elements, are DNA sequences that block the effect of enhancers in a position-dependent manner when insulator-binding proteins are bound to them. This prevents enhancers from stimulating the transcription of the wrong genes on the same chromosome.

transcriptional activators

bind to the upstream and downstream enhancers and are necessary for biologically significant levels of transcription

TFIIs

transcription factors of RNA polymerase 2

TFIID

transcription factor D of RNA polymerase II. This particular TFII recognizes the TATA Box and binds to it.

In the absence of galactose

The GAL80 protein binds to GAL4, and prevents it from transcribing the GAL structural genes