Mutation, Variation, and Gene Expression

What are the two main sources of phenotypic differences

DNA sequence - between species

Gene expression - between cell type

Fixed and lost mutations

Fixed = increase in frequency

Lost = decrease in frequency

Variants

Different versions of DNA sequences that differ by 1+ mutations

Polymorphisms

Mutations that are present in the population in a high frequency (often >1%)

Structural and regulatory mutations

Structural = change in function of a protein

Regulatory = change in expression level of a gene

Single nucleotide variants

Can be transitions (purine to purine) or transversions (purine to pyrimidine)

Tandem repeat mutations

A form of indel (insertion/deletion) occuring between tandem repeats to change copy number. Often arises due to replication slippage or homologous recombination.

Large scale mutations

Duplications, deletions, translocations, inversions, transpositions.

Suppressor mutations

A mutation that suppresses the phenotype of another mutation.

E.g. tRNA mutations

• if a codon is mutated to a stop codon, another mutation in the tRNA anticodon suppresses the original mutation.

Three ways in which small scale mutations arise

Errors during DNA replication and repair

Spontaneous chemical changes of bases that change their pairing

Induced chemical changes of bases from external mutagens

Spontaneous cytosine deamination

Cytosine undergoes deamination to become uracil, this causes C-T transition mutations.

External mutagens - Incorporation of base analogs

Base analogs are non-natural bases that can substitute for natural bases in DNA, they are mutagens if they have unusual base-pairing properties.

External mutagens - specific mis pairing

Some mutagens alter bases in ways that cause mis pairing.

External mutagens - damage that prevents pairing

Benzo[a]pyrene is a component of cigarette smoke that is converted to a mutagen that binds irreversibly to guanine.

Synonymous mutations

Don’t alter amino acid sequence

Conservative mutation

Amino acid is changed to a similar one

Regulatory mutations

Affect the level of gene expression

Deleterious mutations

Compromise organism function - common

Neutral mutations

No effect on organism function - common

Beneficial mutations

Improve organism function - rare

Conservation of DNA

DNA sequences from different organisms having the same sequences, regions of high sequence conservation are likely to be regions important for an organism, mutations here are likely to compromise organism function.

Are mutations induced by unfavourable conditions or are they arising by chance?

They arise by chance, this was discovered by the fluctuation test. E.coli was exposed to bacteriophage, the colonies on each petri plate showed different numbers of resistance, thus proving that they occur randomly during growth.

Promotor and structure

Binds RNA polymerase to initiate transcription. Has a -35 and -10 region with a spacer between them, the length of the spacer is important as RNA polymerase cant bind if it doesnt line up.

Positive regulation of transcription

The activator protein binds the activator binding site, initiating transcription.

Negative regulation of transcription

Repressor protein binds operator, blocking RNA polymerase from binding, no transcription.

The allosteric binding site and transcription

When the effector binds the allosteric binding site, it changes conformation, this can initiate or inhibit transcription

The Lac Operon

When no lactose is present the repressor protein is made from lacI and binds to the operator, no transcription.

When lactose is present it binds to the repressor, stopping it from binding and allowing transcription.

The Oc mutation in the lac operon

A mutation in the operator stops the repressor from binding, causing continuous transcription.

Cis- and trans-acting elements

Cis = act on nearby things

Trans = act anywhere in the cell

What is needed for expression of lactose metabolic enzymes

Lactose presence - negative regulation involves repressors

Glucose absence - positive regulation involves activators

How does glucose involve in the lac operon?

A decrease in glucose causes an increase in cAMP, cAMP binds to CAP which initiates transcription of the lac operon.

What happens when glucose is present and lactose is absent

The operon is off

What happens when glucose is present and lactose is present

The lac operon has little transcription

What happens when glucose is absent and lactose is present

The lac operon is on

Arabinose operon

Positive control - AraC binds araI to initiate transcription

Negative control - AraC changes conformation and binds to activator and operator causing DNA loop which prevents RNA polymerase binding.

Sigma factor

An RNA polymerase subunit that binds to the promoter, bacteria have multiple of these that recognise different DNA sequences

Consensus sequences

Multiple DNA sequences are lined up and the most common nucleotide is taken from that position.

Similarities of gene expression in bacteria and eukaryotes

Both have promotor sequences that bind to RNA polymerase.

Both use activator and repressor proteins.

Differences of gene expression regulation in bacteria and eukaryotes

In bacteria genes are organised in operons (multiple genes under control of a single promotor) but in eukaryotes one promotor controls expression of a single gene which becomes a single protein.

Introns need to be removed before translation in eukaryotes.

In bacteria transcription and translation occur simultaneously, in eukaryotes they occur separately in the nucleus and cytoplasm.

In eukaryotes, DNA is associated with proteins via chromatin, DNA is wrapped around assemblies of histone proteins to form a nucleosome.

Eukaryotes are multicellular so there is division of labour between different cell types.

What is a consequence of DNA associating with proteins via chromatin

It changes the default state of DNA. In bacteria, the default state is on so DNA can always bind RNA polymerase. In eukaryotes, the default state is turned off as DNA is wrapped around nucleosomes, RNA polymerase must get rid of nucleosomes to get to the DNA.

Transcription factors

Regulatory proteins in eukaryotes. They bind specific DNA sequences and act to enhance or block transcription.

The GAL System

This encodes for catabolism of galactose to glucose, there are multiple genes involved, 3 on the same chromosome and one on a different chromosome. GAL4 is the unlinked regulatory TF, it is only expressed in the absence of glucose and binds to upstream activator sequences on each of the genes.

What happens when galactose is / isnt present in the GAL system

No galactose

• GAL4 binds GAL80, preventing transcription

Galactose

• GAL3 binds to galactose, conformational change occurs

• GAL80 binds to GAL3 instead of GAL4

How does GAL4 activate gene expression in the GAL system

GAL4 binds to TFIID which binds to the promotor sequence. GAL4 also binds to the mediator which is associated with RNA polymerase, this helps it to bind to the promotor.

The binding of GAL4 to these allows DNA looping to occur and all factors are brought in close proximity to stimulate transcription.

How can GAL4 act as a tool for synthetic gene control

GAL4 has two domains, DNA binding and activation. The DNA domain can be swapped out for a different DNA binding domain, this allows GAL4 to bind to other sequences and control expression of different genes in response to galactose.

Chromatin and the types

Made of histone proteins, RNA, and DNA that forms chromosomes within the nuclei of eukaryotes.

Euchromatin - open and accessible

Heterochromatin - highly condensed and inaccessible

Four types of chromatin states

Active euchromatin

Inactive (poised) euchromatin - gene is not transcribed due to histone tail modifications

Facultative heterochromatin - can change to euchromatin

Constitutive heterochromatin - permanently heterochromatin

Histone modification

Histone protein tails can be chemically modified via addition of methyl or acetyl groups to specific positions on the tails. These typically occur on lysine residues.

Acetylation is associated with active transcription, methylation is variable.

Chromatin modifiers

Enzymes that can add or remove chemical modifications to histones, they can also add or remove nucleosomes from DNA.

Position effect variegation

Expression of a gene depends on whether the gene integrates into heterochromatin or euchromatin.

Heterochromatin spreading

Heterochromatin protein 1 (HP1) attracts a chromatin modifier that catalyses the heterochromatin histone modifications, spreading heterochromatin. Boundary or insulator proteins block the spread.

GAL activation and chromatin

GAL4 recruits chromatin modifier to the region when activated, this unwinds DNA from nucleosomes allowing RNA polymerase to bind and initiate transcription.

DNA methylation

Typically methylated on C bases on CG dinucleotides. CG are found mostly on promotors, if they are methylated, transcription is repressed.

DNA methylation can be inherited following replication, enzyme recognises hemi methylated DNA and methylates the opposite C base.

Small RNA regulation of gene expression

Small RNA molecules repressing translation as they form complementary base pairs with the mRNA.

A dicer complex cuts double stranded RNA into small single stranded RNA, RISC binds small RNA, denatures it and incorporates another strand. RISC finds complementary mRNA and binds it to prevent translation via degradation.

Small RNA with perfect complementarity - siRNAs, they act with RISC to cleave mRNA

Small RNA with partial complementarity - miRNAs, they act with RISC to inhibit translation of mRNA

Alternative splicing

Spliceosome splices out introns, though it does not always know which is the correct splice site. Sometimes exons are included and sometimes they are skipped.

Who discovered transposons and how?

Barbara McClintock in the 1940s discovered that alleles in maize changed quickly. One unstable mutant had segments of lost colour die to chromosome breakage (Ds), one would revert to wild type colour at high frequency. Reverting to WT was due to Ds transposing into the C gene, inactivating it, when Ds jumped out purple spots appeared.

Transposons

DNA sequences that catalyse their own movement in the genome.

Properties of transposons

Encode transposase enzymes which catalyse transposition.

Have inverted or direct repeats at termini

What are the two bacterial classes of transposons

Simple - single transposons with short inverted repeats at termini, they are called insertion sequences.

Composite - have insertion sequences at each terminus with genes between them, when they move they move as a whole (genes included), these are called transposons.

Direct repeats and how are they created

Direct repeats are DNA sequences repeated somewhere else on the same strand, they are created by transposons when they move to a new site. When the transposon jumps it makes staggered cuts in the DNA and inserts itself, these staggered cuts are then filled in my DNA polymerase, creating direct repeats.

Inverted repeats

Repetition of the same DNA sequence 5’ to 3’ on opposite strands. These are recognised by transposase enzymes and define the ends of a transposon. The enzyme binds to these repeats and cuts out the transposon to move it elsewhere.

Three classes of transposon defined by how they move

Helitrons

Class I - Retrotransposons, transpose through RNA intermediate, they are transcribed into RNA and reverse transcribed into DNA. The DNA copy inserts back into the genome

Class II - moved by a DNA intermediate, DNA is moved to a new site.

Conservative vs replicative transposons

Conservative - move by cutting themselves out of DNA and re-inserting at a new site. Cut and paste.

Replicative - move by copying themselves, copy and paste. These can dramatically increase in number.

Autonomous vs non-autonomous transposons

Autonomous - encode their own transposase gene and can catalyse their own transposition.

Non-autonomous - rely on transposases made by autonomous. Usually more of these in the genome