Genetics Exam 4

Transposable elements

jumping genes, defined ends and encode the functions responsible for their own movement

Class I elements (retrotransposons)

RNA as the mobile intermediate, often the remnants of a virus that integrated into genome, only in eukaryotes, code for reverse transcriptase (pol), copy and paste mechanism, very common in mammalian genomes

Class II elements

DNA as the mobile intermediate, smallest and simplest DNA transposon, found in bacteria & eukaryotes, encodes a transposase enzyme, sequences at either end form an inverted repeat

Reverse transcriptase (pol)

enzyme that can convert RNA to DNA

Copy and paste mechanism (class I)

the element that moves is a copy of the transposon that is still present in another location

Transposase enzyme (class II)

catalyzes the excision and insertion of the element to another location in genome

Inverted repeat (class II)

the sequences are either end of the insertion sequence so that the ends read the same 5’ to 3’, this allows element to form hairpin structure by intra-strand base pairing

Transposable elements (TE) and evolution

TE’s are a principle source of mutation, genetic variation, and evolution, ex. telomerase

Vertical gene transfer

when genetic material is passed from parents to offspring, mutations do occur but the amount of variation is slight, genetic content of offspring very similar to parents

Horizontal gene transfer

individuals in the same generation can exchange DNA, can transmit a little or a lot of genetic info between different lineages, horizontally-acquired DNA can then be transmitted vertically to offspring of recipient

Direction of horizontal gene transfer

DNA is transferred uni-directionally from a donor to a recipient, if the recipient integrates DNA into genome it may express those phenotypes nd transmit to future offpsring

Horizontal gene transfer in bacteria

occurs in plasmids, many are circular, vary in size and gene composition, transmitted across generations

Horizontal gene transfer disadvantages

1. transcripts can be harmful

2. insertions can disrupt gene function

3. metabolic cost to replicate acquired DNA if new genes not providing selective advantage

Types of horizontal gene transfer

transformation, conjugation, transduction

Transformation

uptake of DNA by a cell, competent cells can take up extracellular DNA from the environment, replicate, then remain a plasmid or be incorporated into genome - need 2 crossover events

Selectable markers

used to identify competent cells that have taken up the plasmid

Conjugation

transfer of DNA from one cell to another through direct cell contact, mediated by transfer genes encoded in donor cell’s genome, can be between bacteria and eukaryotes

Transduction

the introduction of foreign DNA from a host cell into a recipient cell mediated by a virus

Structure of viruses

genome encased in a protein capsid, can be single or double stranded, either DNA or RNA, many different pathways for expression

Bacteriophages

viruses that infect bacteria

Life cycle of lytic phage

Phage attaches to the surface of the bacterium, phage DNA is injected into the host cell, host cell makes new phage proteins and host cell DNA is degraded, new phage particles assemble inside the host, the host cell is lysed and new phage are released

Generalized transduction

at the stage when new phage particles assemble inside host: phage acquire fragments of host DNA, when phage infects new host DNA from the previous host is transduced, sometimes only host DNA not virus’ own genome

Life cycle of lysogenic phage

two possible life cycles once it infects host - lytic or lysogenic, the phage inserts its genome into the host’s chromosome (prophage), can replicate as part of the chromosome, can remain integrated indefinitely or at any time can be excised and initiate lytic cycle

Specialized transduction

when the prophage is excised from the host genome sometimes it is cut out imprecisely and parts of the host genome become part of the phage, pieces of host genome can be carried to next host

Antibiotics

medicines that kill or limit the growth of bacteria

CRISPR

clustered interspaced short palindromic repeats, system bacteria use to recognize foreign DNA (immune response), laboratory use to cut DNA sequence wherever we want

CRISPR structure

CRISPR associated genes (Cas genes), leader = promotor, repeats with spacers in between, bacteria remembers infection when virus infects bacteria a part of the viral genome is added in place of a spacer

gRNA

used in laboratory use, the crRNA and tracrRNA as one molecule

PAM site

recognizes target site and where to cleave target DNA sequence

Limitation of CRISPR/Cas9 in human disease

difficult to deliver to mature cells in large numbers, some issues with reactions to delivery methods, some cells that take in CRISPR/Cas9 might not have genome editing activity, not 100% accurate

Ethical concerns of CRISPR/Cas9

editing somatic cells vs germ line cells, germ line edits would be passed onto next generation, designer babies

Gene expression: Bacteria

regulation of gene expression occurs primarily at the initiation of transcription, transcription start site (TSS) is before translational start site, upstream and downstream regions

Gene expression: Eukaryotes

regulation of gene expression occurs primarily at the pre-initiation of transcription, but also involves RNA splicing, translation, other non-coding RNAs, and microRNAs, RNAP is recruited to the promotor by general transcription factors

RNA polymerase

enzyme complex that transcribes DNA into RNA, template stand is where RNA polymerase reads and transcribes, animals have RNAP I, II, III, plants have those three plus IV, V, different RNAPs transcribe different types of genes

Upstream region

upstream of the gene contains signals needed for the initiation of transcription, RNA polymerase binds

Downstream region

may contain other regulatory region (transcript stability, translation)

Stages of transcription

Initiation, elongation, termination

Initiation

core promoter of many genes in eukaryotes include the TATA box, the TATA binding protein (TBP) binds to the TATA box and facilitates the binding of other TFs and RNAP which creates the pre-initiation complex

Initiator element

if gene does not have TATA box another promoter sequence the TBP binds to

5’ untranslated region (UTR)

the region of transcript between the TSS and the start of translation, there is also a 3’ UTR, these regions contain regulatory sequences

Sigma subunit of RNAP

in bacteria one of RNAPs multiple subunits, binds to DNA and recruits RNAP, different sigma factors bind to different core promoters and regulate transcription of different genes

General transcription factors

bind to core promoter, common among genes, initiates transcription

Sequence specific transcription factors

known as transcription factors, binds to specific DNA sequences to regulate specific gene expression (time, tissue, how much)

Consensus sequences

binding sites for a particular TF can sometimes vary by a bp or two, thus binding sequences often represent by consensus sequences, size of the letter indicates how important it is in the binding sequences

Master regulator TFs

regulate the expression of other TF genes

Effector TFs

regulate the expression of structural proteins and enzymes

TF binding sites

commonly upstream but can be in the first intron or downstream

in vivo vs vitro binding

in vivo = within a living organism, in vitro = outside of a living organism (test tube or petri dish)

Activators

TFs that turn transcription up or on by binding to enhancer sequences in the DNA

Repressors

TFs that turn transcription down or off by binding to silencer sequences in the DNA

Cis-regulatory module (CRM)

binding sites for TFs clustered upstream of the TSS

TF expression

a number of TFs will have binding sites in a CRM but only certain binding sites will be occupied based on each cell type which expresses different TFs

Transcriptional reporter

in core promoter, where and when the gene is transcribed?

Translational reporter

at the end of the gene, where and when is the protein found?

Hox genes

master regulators because they regulate expression of many other genes, they can turn on developmental programs, mutations in hox genes results in changes in mutations that leads to phenotypic mutations, can target different effector genes

DNA binding domain

where the TF interacts with the DNA sequence, specific for each TF, the amino acid sequence and structure determine the DNA binding site specificity, TFs classified by their DNA binding domain

ChIP

used to find locations where TFs are bound to DNA

1. extract chromatin with transcription factor from cells

2. fragment chromatin and add specific antibody

3. precipitate complex using antibody

4. sequence DNA and map genome to find binding site

5. the sequence nearby the binding site is surveyed for genes and other functional elements

Open vs closed chromatin

closed chromatin resistant to TF binding, open chromatin available for TF binding, pioneer TFs can bind to closed chromatin

Histone modifications

the core four histones have a tail of amino acid that interacts with other proteins and become modified, commonly H3, many modification appear at lysine (K), sometimes serine (S), threonine (T), or arginine (R)

Histone code

the specific combinations of histone modifications at a particular part of the chromatin, different locations upstream of and within the gene often have particular modifications, not every nucleosome at that site may be modified in the same way

Types of histone modifications

methylation, acetylation, phosphorylation, ubiquitination (ubiquitin protein added to histone protein)

Epigenetics

heritable change in phenotype without underlying change in DNA sequence, due to histone modification, some modification can be added or removed, but some are permanent when they are inherited when a eukaryotic cell divides by mitosis, can also be inhertied across generations from parent to child

Mediator complex

a large complex of 30 evolutionarily conserved proteins, forms a bridge between TFs bound to CRM and RNAP II

Elongation

RNA polymerase moves along the DNA template strand making a RNA chain

Timing of transcription

the time it takes to transcribe a gene has implications for how quickly organisms can respond to their environment, bacteria transcribed much faster than eukaryotes

Transcript

RNA molecule that is made from the gene, short lived because RNA is unstable

Termination

the process of ending transcription

Operon

in bacteria genes with a similar function are transcribed together and controlled by the same promoter

Termination in bacteria: Rho-dependent

an RNA binding protein called Rho binds to a 70-100 bp region called rut, dissociates the RNA from the DNA

Termination in bacteria: Rho-independent

the transcript has an inverted repeat structure downstream of the stop codon, when the repeat forms a hairpin structure through intra-strand base pairing transcription terminates

Termination in eukaryotes

RNAP I (ribosomal genes) - similar protein to Rho involved, RNAP II (most transcripts) - the polyadenylation sequence is recognized by factors which recruit proteins to continue and complete RNA processing, RNAP III - termination factor is idependent, a stretch of thymines

RNA processing eukaryotes

RNA modified after transcribed, cap added to the 5’ end for nuclear export and translation, PolyA tail added to 3’ end for stability, introns removed, exons spliced together = mRNA (mature)

Alternative splicing

general term for more than one mRNA is made from precursor mRNA (pre-mRNA) because different sequences in the RNA are used as the splice donor and acceptor sites

Spliceosome

the macromolecule complex made up of RNA and proteins that carries out splicing

Types of alternative splicing

exon skipping, alternative 5’ site, alternative 3’ site, mutually exclusive exons

Messenger RNA (mRNA)

transcripts that are translated into proteins

Ribosomal RNA (rRNA)

RNA that together with proteins form the ribosome

Transfer RNA (tRNA)

RNA involved in translating the mRNA sequence into amino acids, brings amino acids during translation, anticodon:codon binding, extensive intrastrand base pairing creates specific loops

Non-coding RNA

long ncRNAs, small nuclear RNA snRNA, tRNAs, rRNAs, microRNAs

Long ncRNAs

affect translational initiation, have diverse functions many not known yet

Small nuclear RNA (snRNA)

base pair with pre-mRNA to direct splice site choice

rRNAs

structural components, interact with proteins, base pair with mRNA, with each other, etc.

microRNAs

base pair with mRNA to prevent translation, around 22 bp, form a double stranded RNA (dsRNA) with a specific target in the mRNA, complementary base pair binding, target sites in animals are in the 3’ UTR, target sites variable in plants, sometimes multiple sites or just one, mRNA bound to microRNAs is either targeted for degradation or cannot be translated

RNAi

an experimental application of the role that microRNAs play naturally, used to turn genes off, after one strand is degraded siRNA (short interfering RNA) pairs to mRNA target

Network modules

few interactions among TFs many targets per transcription factor = processes that require an immediate response, many interactions among TFs few targets per transcription factor = processes that occur at specific times need tight control

Translation bacteria

mRNAs are translated as they are being transcribed, no nucleus

Translation eukaryotes

transcription and translation occur at different times and locations

Anticodon

binds to mRNA and is the adaptor between RNA and amino acids

Aminoacyl synthetases

a family of enzymes responsible for loading the amino acids on the tRNA, each recognizes the anticodon of the tRNA and attaches correct amino acid at the 3’ end

Ribsome

pockets in the structure have different roles, A - aminoacyl site, P - peptidyl site, E - exit site, mRNA threaded between ribosomal subunits

3 phases of translation

initiation, elongation, termination

Initiation translation

1. mRNA binds to small subunit of ribosome at the ribosome binding site (rRNA), mediated by initiation factor proteins

2. Initiator tRNA (with f-met) binds to start codon

3. large subunit then binds to complete initiation

4. Initiator tRNA is in P site, A and E are empty

Elongation translation

1. A tRNA binds to the codon in the A site

2. Amino acids of tRNA in P and A are placed in the ribosome’s active site → peptide bonding

3. ribosome translocates down the mRNA by three nucleotides

4. a new mRNA codon opens at A

5. if there is a tRNA in the E site translocation ejects it

6. the process repeats down the mRNA length

Termination translation

1. the A site encounters a stop codon, no tRNA can pair with stop codon

2. a release factor protein binds to stop codon, breaks bond of tRNA in the P site with the polypeptide, ribosome subunits dissociate, mRNA separates

Regulation in translation

likely occurring at the initiation of translation, involves a specific interaction between the mRNA sequence and proteins at the 5’ or 3’ UTR

Degeneracy

multiple codons code for the same amino acid

Wobble

unconventional base pairing between the third base of the codon with the first tRNA anti codon, responsible for degeneracy

Evolutionary accepted mutations

mutations in the DNA that result in different amino acids (missense) and are observed in nature, do not have big effect on the function of proteins

Dayhoff substitution matrix

orthologous (evolved from common ancestor gene) proteins are aligned, compares how often each amino acid is conserved, determines that some substitutions are more common than others

Universality of genetic code

further indication that all existing life can be traced back to a common ancestor