Genetics Exam 3

Negative supercoiling

The chromosomal DNA in bacteria is negatively supercoiled.

Negative supercoiling has two major effects:

1. Helps in the compaction of chromosome

2. In localized regions, creates tension that may be released by DNA strand separation

positive supercoiling

Positive supercoiling serves to compact DNA, regulate gene expression and coordinate the transcriptional response to environmental signals.

DNA gyrase

DNA gyrase introduces negative supercoils using energy from ATP, can relax positive supercoils, untangle intertwined DNA .

DNA topoisomerase I

relaxes negative supercoils

Eukaryotic chromosomes

Each chromosome contains a single, linear molecule of DNA, contain multiple origins of replication

Centromeres

regions that play a role in segregation of chromosomes

telomeres

specialized regions at the ends of chromosomes

composition and function of a nucleosome

DNA is packaged around histone proteins to form nucleosomes. Within the chromatin and composed of a double-stranded segment of DNA wrapped around an octamer (composed of two copies each of four different histone proteins), associate with each other to form a more compact structure.

Histone proteins

basic, contain many positively-charged amino acids (lysine and arginine), bind to the negatively-charged phosphates along the DNA backbone

Five types of histones

H2A, H2B, H3 and H4 are the core histones

▪ H1 is called the linker histone (binds to DNA in the linker region, less tightly bound to DNA than core histones, helps to organize adjacent nucleosomes)

euchromatin

less condensed regions of chromosomes, transcriptionally active, loop domains are less compacted.

heterochromatin

tightly compacted regions of chromosomes, transcriptionally inactive, loop domains are more compacted.

constitutive heterochromatin

regions that are always heterochromatic, permanently inactive for transcription, and usually contain highly repetitive sequences.

facultative heterochromatin

regions that can interconvert between euchromatin and heterochromatin.

cohesin

a multiprotein complex that also contains SMC proteins SMC1+SMC3, promoting binding between sister chromatids during mitosis and meiosis, only found at the centromere and degraded during anaphase.

condensin

compacts chromosomes during mitosis and meiosis. Both condensin I and II facilitate the reorganization of chromosomes into radial loop arrays.

transposition

involves the integration of small segments of DNA into a new location in the genome.

Small, mobile DNA segments are termed transposable elements (TEs) - Barbara McClintock.

simple transposons

Used widely by transposable elements, or transposons, TE is removed from its original site and transferred to a new target site.

Mechanism is called a cut-and-paste mechanism because the element is cut out of its original site and pasted into a new one.

direct repeats (DRs)

all TE are flanked, which are identical base sequences that are oriented in the same direction and repeated.

insertion element

simplest TE, which is flanked by inverted repeats

inverted repeats

DNA sequences that are identical but run in opposite directions

transposase

catalyzes the transposition event

autonomous elements

contain all the information necessary for transposition or retrotransposition

nonautonomous element

lacks a gene such as one that encodes transposase or reverse transcriptase, which is necessary for transposition.

retrotransposons

move via an RNA intermediate and are transcribed into RNA, found only in eukaryotic species.

use an RNA intermediate in their transposition mechanism.

LTR retrotransposons

are evolutionarily related to known retroviruses, contain long terminal repeats (LTRs)

LTR retrotransposon movement requires two key enzymes:

Reverse transcriptase uses this RNA as a template to synthesize a double-stranded DNA molecule.

Integrate recognizes LTRs at the ends of the DNA, makes cuts at a target site in the host chromosome, and catalyzes the insertion of the TE into the site

Non-LTR retrotransposons

do not resemble retroviruses in having LTR sequences, moved by target-site primed reverse transcription.

Meselson’s and Stahl’s

by using a density-labeling technique that showed DNA replication was semi-conservative.

DNA helicase

binds to the origin, breaks the hydrogen bonds between the DNA strands, further separates the DNA strands, composed of six subunits, travels along the DNA in the 5’ to 3’ direction, uses energy from ATP

Primase

short RNA primers being synthesized

DNA polymerase

are the enzymes that catalyze the attachment of nucleotides to synthesize a new DNA strand.

DNA pol III

normal replication, responsible for most of the DNA replication, processive enzyme due to several different subunits in the DNA pol II holoenzyme, synthesizes a daughter strand of DNA

DNA pol I

normal replication, removes the RNA primers and replaces them with DNA, uses a 5’ to 3’ exonuclease activity to digest the RNA and 5’ to 3’ polymerase activity to replace it with DNA

DNA ligase

catalyzes the formation of a covalent (ester) bond to connect the DNA backbones, covalently links the DNA backbones in the Okazaki fragments together

leading strand

one primer is made at the origin, DNA pol III attaches nucleotides in a 5’ to 3’ direction as it slides toward the opening of the replication.

Lagging strand

synthesis is also in the 5’ to 3’ direction, away from the replication fork; many RNA primers are required; DNA pol III uses the RNA primers to synthesize small DNA fragments.

Explain how DNA polymerase III attaches nucleotides to a growing DNA strand while also decreasing the error rate during replication.

Explain why telomerase is needed for replication of the ends of eukaryotic chromosomes.

1. Binding 2. Polymerization 3. Translocations are steps repeated many times to preserve the length of one strand.

silent mutations

base substitutions that do not alter the amino acid sequence of the polypeptide

missense mutations

base substitutions in which an amino acid change does occur

nonsense mutations

base substitutions that change a normal codon to a stop codon

frameshift mutations

addition or deletion of a number of nucleotides that is not divisible by three

Describe how a mutation within the coding sequence of a gene may alter a polypeptide’s structure and function.

Intragenic

suppressors a second mutation in the same gene restores function

Intergenic

a different gene that counteracts the detrimental effects of a mutation in a first gene

depurination

the removal of a purine (guanine or adenine) from the DNA to form an apurinic site, which can be repaired by DNA repair pathways.

deamination

the removal of an amino group from the cytosine base; C to U/T mutations, can be repaired by DNA repair pathways

Tautomeric shift

a temporary change in base structure; the common, stable form of thymine and guanine is the keto form (enol); the common, stable form of adenine and cytosine is the amino form (imino)

Base excision repair (BER)

involves a category of enzymes known as DNA N-glycosylases (eliminates abnormal bases)

Nucleotide excision repair (NER)

type of system can repair many types of DNA damage, including thymine dimers and chemically modified bases, missing bases, some types of crosslinks

mismatch repair system

recognize and correct a base pair mismatch; DNA polymerases have a 3’ to 5’ proofreading ability that can detect base mismatches and fix them

Specific to the newly made strand (occurs right after replication)

In E. coli, three proteins, MutL, MutH and MutS detect the mismatch and direct its removal from the newly made strand

MutH distinguishes between the parental strand (methylated) and the daughter strand (not methylated)

Promoter

DNA site for RNA polymerase to bind; signals start of transcription

Terminator

DNA site signaling end of transcription

activator

bind to DNA and increase transcription (positive control)

repressor

bind to DNA and inhibit transcription (negative control)

mRNA

the "messenger" carrying genetic code from DNA to the ribosome

rRNA

the structural and catalytic component of the ribosome

tRNA

the "transfer" molecule that brings specific amino acids to the ribosome

inducible

Those that increase transcription of inducible genes are termed inducers; May bind to activators and cause them to bind to DNA. May bind to repressors and prevent them from binding to DNA. (cAMP-CAP, lac operon)

Those that inhibit transcription of repressible genes include:

Corepressors which bind to repressors and cause them to bind to DNA.

Inhibitors which bind to activators and prevent them from binding to DNA. (trp operon)

lactose is absent

a repressor protein binds to the operator, blocking transcription

lactose is present

it acts as an inducer by binding to the repressor, causing it to release and allowing transcription to occur

catabolite activator protein (CAP)

• Glucose levels are regulated by the catabolite activator protein (CAP); when glucose is low, CAP binds to the CAP site and stimulates transcription, ensuring the bacteria uses lactose only when glucose is scarce.

trp repressor

When tryptophan levels are low, tryptophan does not bind to the trp repressor, preventing the repressor protein from binding to the operator site.

attenuation

Another mechanism of regulation is attenuation. When attenuation occurs, the RNA is transcribed only to the attenuator sequence, and then transcription is terminated

GTFs

bind to DNA and help position RNA polymerase to begin the process of creating an RNA copy of a gene (transcription).

TFIID

Composed of TATA-binding protein (TBP) and other TBP-associated factors (TAFs); Recognizes the TATA box

TFIIA

Binds to TFIID; Promotes TFIID binding to the TATA box

TFIIB

Binds to TFIID; Enables RNA pol II to bind to core promoter; Also promotes TFIIF binding

TFIIF

Binds to RNA pol II; Plays role in RNA pol II’s ability to bind TFIIB and the core promoter; Plays role in ability of TFIIE and TFIIH to bind to RNA pol II

TFIIE

Role in the formation or maintenance (or both) of the open complex; May exert effects by facilitating binding of TFIIH to RNA pol II and regulating the activity of TFIIH

TFIIH

Multisubunit protein; Multiple roles

Identify the functions of the components of the pre-initiation complex for eukaryotic transcription.

1. GTFs

2. RNA Pol II - enzyme, catalyzes linkage of nucleotides in the 5’ to 3’ direction, using DNA as the template

3. Mediator - Multisubunit complex; Mediates effects of regulatory transcription factors on RNA pol II; Subunits vary depending on cell type and environmental conditions; Plays key role in RNA pol II switch from initiation to elongation

5’ cap

Recognized by cap-binding proteins which play roles in:

1. Movement of RNAs out of the nucleus

2. Early stages of translation

3. Splicing of introns.

PolyA tail

NOT coded in the gene sequence; Added enzymatically after the gene is completely transcribed; A long PolyA tail is important for mRNA stability, Export from the nucleus, Synthesis of polypeptides.

Introns

(intervening sequences) are removed

Exons

(coding sequences) are connected together

Presence of splicing factors

exons that are recognized and joined together, resulting in one or more specific splice variants, that bind to the spliceosome (a complex of RNA and proteins).

absence of splicing factors

outcome is unpredictable; a different set of exons may be included or excluded, leading to a different splice variant and the spliceosome may bind to incorrect splice sites or not assemble at all.

start codon

AUG (which specifies methionine) = start codon

• Defines the reading frame for all following codons

  • Example: 5’ - AUG CCC GGA GGC ACC GUC CAA U - 3’

    • Met - Pro - Gly - Gly - The - Val - Gln

stop codons

UAA, UAG and UGA = termination, or stop codons

• Example: GGU, GGC, GGA, and GGG all code for glycine (are synonymous)

Translate a mature mRNA sequence into a polypeptide chain (using a provided codon table)

four levels of structure in proteins

• Primary - amino acid sequence linked by peptide bonds

• Secondary - the primary structure of a protein folds to form regular, repeating shapes (a helix, b sheet); stabilized by the formation of hydrogen bonds between atoms located in the polypeptide backbone.

• Tertiary - the short regions of secondary structure in a protein fold into a three-dimensional polypeptide; Structure determined by hydrophobic and ionic interactions as well as hydrogen bonds and van der Waals interactions.

• Quaternary - proteins made up of two or more polypeptides associate with one another to make a functional protein

Calculate the expected percentage of a radiolabeled amino acid from a cell-free translation experiment

Describe the functions of the 16S, 30S, and 50S subunits of the bacterial ribosome, identifying any key sites within them.

16S - 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, decoding function. codon-anticodon recognition, Shine-Dalgarno binding

30S - decoding, initiation, mRNA binding (A site, P site)

50S - peptide bond formation, elongation, peptide exit (peptidyl transferase, E site, exit tunnel) 

Explain what’s happening in the ribosome during the elongation stage of translation.

1. A charged tRNA brings a new amino acid to the ribosome so that it can be attached to the end of the growing polypeptide and binds to the A site

2. Next step of elongation is peptidyl transfer—the polypeptide is removed from the tRNA in the P site and transferred to the amino acid at the A site

3. The ribosome then moves, or translocates, to the next codon in the mRNA, moving the tRNAs at the P and A sites to the E and P sites

4. Uncharged tRNA is released from the E site.

5. Repeat steps 1-4 until a stop codon appears