BIOB11 Final Exam Review

Alternative splicing

Alternative splicing

How different exons in eukaryotes are included and lead to isoforms all coming from same genes with different functions, sizes, and structures. Introns are much larger than exons and the genes can vary in size.

Lariat

In splicing, each intron removed forms a lasso.

Intron ends are joined together and exons joined together.

snRNA

in splicing, this does base pairs with 5’ splice site, 3’ splice site and a branch point in the intron. Splicing and binding is coordinated with consensus sequence.

snRNPs

A complex of the 5 snRNAs (U1, U2, U4, U5, U6) with the proteins, and together they form a spliceosome. Assemble on mRNA molecules.

U1 snRNP

binds to 5’ splice site

U2 snRNP

binds to branch point site, and adenosine bulges out

U4 U6 pair and U5 snRNPs

enter and 6 replaces 1 at 5’ splice site. 4 and 1 are released.

Exon junction complex (EJC)

Marks newly completed splicing event

Splicing errors

Changing sequences included in mRNA. Skipping an exon. May cause diseases.

Splicing error consequences

Mutations prevent excision or alter splicing, change stability of RNA, RNA degradation, accumulation of unspliced intermediates, frameshift mutations, missense mutations, impaired mRNA transport.

Impaired mRNA transport

Pre-mRNA is mostly not matured. mRNA is rapidly degraded and turned over. Huge amount of RNA not translated. Needs a process to get rid of unprocessed pre-mRNA.

Default fate of RNA exonuclease: cleave it up.

Nuclear pore complex

Get help out of nucleus into cytoplasm. Only occur if right set of proteins are attached to the RNA molecules.

pre-mRNA proteins

RNA polymerase, guanyl transferase, poly(a) polymerase, U1 snRNP

rDNA

Found in clusters of nucleoli, ribosomal biosynthesis, and other noncoding RNA.

S-value

Measurement of sedimentation rate during centrifugation, affected by mass and shape. The larger the S value, the larger the molecule, the faster the sedimentation.

rRNA

Multiple copies in genome. No amplification step makes it more advantageous to have multiple of. Small (RNA pol 3) and large (RNA pol 1) subunits.

Precursor: methylated and cleaved during transcription.

rRNA modifications

Isomerization (uridines processed to become pseudouridine)

Methylation (methyl group to 2’OH of ribose)

snoRNAs made by RNA pol 3

tRNA

80 nucleotides in length, marked by which amino acid it carries. Transcribed by RNA pol 3.

Anticodon loop at one end, and 3’ amino acid acceptor arm on opposite end.

Many, does not amplify, more the merrier, process goes faster.

tRNA modifications

Carry amino acid based on anticodon sequence. Tertiary structure twists into an L-shape.

Non-overlapping code experiment

Mutating one C and seeing if it affected three amino acids or only one. A single nucleotide change results in one amino acid change. Conclude it is non-overlapping.

Reading frame

Three possible based on where ribosome starts.

Determined by AUG - Methionine - Met.

Degenerate code

More than one code can code for same amino acid, creating redundancy.

Differ mostly in third letter (wobble base pairing)

Grouped with having similar amino acid properties.

Silent mutation

Alters the nucleotide sequence, but encodes for same amino acid

Missense mutation

Changes one amino acid

Nonsense mutation

Mutation that leads to a stop codon.

Wobble-base pairing

U can pair with A or G. G can pair with U or C. I can pair with U, A, or C. First two in codon are set, third base pairing can differ.

Charging tRNA

The appropriate tRNA must be covalently linked to the free OH on the adenosine at the 3’ end of the tRNA (amino acid acceptor stem).

Aminoacyl-tRNA synthetase

Catalyzes the charging on tRNA with amino acid. Interacts with anticodon loop and amino acid acceptor arm. Modification in both regions.

tRNA is binded to tryptophan and the amino acid addition is catalyzed.

Has a proofreading function (pushes AA into site and breaks bond)

Translation

Amino acids are added to the C-terminal end of growing polypeptide chain.

Ribosome has A(minoacyl), P(eptidyl), E(xit). Small ribosomal subunit helps decode mRNA by interacting with anticodon. Large ribosome aids in peptide synthesis by interacting with aminoacyl-tRNA and catalyzing bond formation.

Ribozyme

Ribosomes that have catalytic activity (ex. spliceosomes)

Polypeptide structure

Linear sequences of amino acids, linked by peptide bonds, which join the carboxyl end of one to the amino of another. Differs by side chain/R-group

Functional domains

Proteins fold into these, and they have a particular function. Fold independently, with particular function.

Domains attributes

Different proteins may have same domain

Domains are shuffled and used in different cells

It is the function unit and structural unit

Same domain doesn’t mark difference in sequence

In one polypeptide, you could have multiple domains

DNA binding protiens

Contain a common set of domains, and this is a limited set of domains. We can move sequences like with exon shuffling. This is how the same can be found in multiple different proteins.

Initiation

Met is start codon, determines reading frame. IFs/eIFs are required.

Prokaryotic initiation

Shine dalgarno sequence helps position ribosome to correct AUG.

IF1: attach mRNA, IF2: GTP-binding proteins help attach AA-tRNA, IF3: prevent premature attachment of large subunit.

N-formylmethionine (fMet)

(prokaryotes) The first trna that gets positioned and binded in P site of ribosome. Once initiator tRNA is found, we release initiator proteins and GTP hydrolysis occurs so large subunit can bind.

Eukaryotic initiation

Happens once ends are matured. eIFs require more protiens (12)

Kozak sequence. GTP bound to eIF2 is hydrolyzed and is released with rest of eIFs.

eIF1 and 1A

Conformational change to allow for binding of mRNA

eIF3

will interact with eIF4G on mRNA complex

eIF4E

binds to 5’ cap

eIF4A

has helicase activity that uses ATP hydrolysis to unwind any double stranded regions in mRNA

eIF4G

links 5’ cap and 3’ poly A tail, which converts mRNA into a circular message

Kozak Sequence

Small ribosomal subunit + eIFs find end of mRNA scans along until reaches 5’-CCACCAUGC-3’). Differing sequence may effect the proteins produced because they will have different amino acid sequences at the N-terminus. Eukaryotes.

Shine-Dalgarno sequence

AUG guided by a specific sequence of nucleotides upstream (5’-AGGAGG-3’). It is complementary to a sequence near the 3’ end of 16s rRNA, which positions the ribosome in the correct spot. Prokaryotes.

Elongation

A new peptide bond forms between amino group of incoming amino acid and C-terminus of growing chain.

AA-tRNA synthetase brings to empty A site, Peptidyl transferase catalyzes new peptide bond, large subunit moves first, oldest tRNA ejected

Transcription elongation factors

We need a certain level of accuracy. These hydrolyze GTP to provide energy for conformation changes. EF-Tu is part of an accuracy check that corrects tRNA base pairing.

Elongation accuracy check

(EF-Tu) Small subunit hydrogen bonds with codon-anticodon. A correct pairing will trigger conformational changes in ribosome. Stuff is moved away until proofreading function moves it close enough to bind.

Termination

Stop codon signal end. N to C direction. Once it reaches stop codon in A site, there is no corresponding tRNA. A release factor enters and instead of a polypeptide bond, a water molecule is added (-CO-OH), catalyzes release.

Pluripotent stem cells

Produce all cell types in body (embryonic stem cells)

Multipotent stem cells

Produce a related group of cell (hematopoietic stem cells)

Unipotent stem cells

Only produce cells of their own type but have property of self renewal required to be labelled as a stem cell.

In situ hybridization

mRNA is detected through base-pairing with a labeled probe, complementary to a specific sequence in the RNA of interest. Confirmed that expression can be restricted to specific sites in an organism.

Genomic equivalence

How it is possible that every somatic cell contains the same DNA. The idea that the cell does not throw away its DNA.

2D gel electrophoresis

Used to separate proteins by charge and by mass. Brain v. Liver have similar sections light up, but still many are different. Gives a general look.

RNA sequencing

Get a bunch in a cell. All the mRNA is analyzed. Can study transcriptome (all transcripts of RNA) and find genes that differ in samples. Tell what is highly transcribed by relating it to a genome.

Fragment RNA → reverse transcription → PCR → sequence.

Constitutive expression

Genes coded in all cell types, all the time.

Beta-actin gene

Exhibits constitutive expression. Cell function requires right genes to be expressed (right cells, right time, right amount).

Heatmaps

Displaying groupings (treatments being compared or cluster'/subtypes based on similar expressions of genes) and a colour scheme reflects level of expression. Each line represents a different gene that was expressed.

Cis-regulatory elements

Act on same strand of DNA they are found on. Sequences encoded on the same chromosomes as the gene they affect. Are the same in each cell type, TFs are what differ.

Consensus sequences. Promoters, enhancers, silencers.

Trans-regulatory elements

Can be expressed from any chromosomes. They contain structural motifs that recognize/bind to cis regulatory elements. Many different one work with many cis ones.

Transcription factors. Activator, Repressor.

ChIP-seq

Chromatin immunoprecipitation and sequencing. Cross-linking, break up, antibody binds and pulls away from everything else in the cell, sequencing.

Transcription factors (TFs)

Proteins that bind specific DNA sequences and influence transcription. Forming complexes with other trans-regulatory elements. Everything that binds to cis-regulatory elements.

A single gene can be regulated by multiple TFs. A TF can be involved in regulating multiple genes. A TF can have different effects in different contexts. A single gene can be regulated by multiple cis-regulatory elements.

Binding domains

Stuff often binds as a dimer, and they can now recognize adjacent groups and double how much they can check out. Sometime homo or heterodimers (working with greater numbers).

Promoters

Sequences of DNA where RNA polymerase can be recruited to initiate transcription

Enhancers

Tells how much gene product is made from a promoter. One gene may have multiple (the one being used is what changes a situation).

Studied with GFP, deletion mapping, and reporter gene fusion.

Silencers

DNA regulatory sequences that prevent promoter use and inhibit transcription. Restricts gene expression to its proper cell and time. Tests so certain genes are not expressed

Green fluorescent protein (GFP)

Beta-galactosidase; fix and stain embryo for galactosidase activity. DNA sequences are fused to reporter gene.

Deletion mapping

Deleting various segments to see was in transcription is affected, and if you delete an enhancer.

Reporter gene fusion

Occurs when we fuse one strip of genes with reporter genes and track what cis-regulatory elements make the strip important.

Activator

Binds enhancer DNA elements

Repressor

Binds silencer DNA elements

Nucleosomes in TF binding

Some TFs need to destabilize nucleosomes, as their binding sections are facing towards histones. Major groove is used as a binding site. Activators can affectl chromatin structure in order to get to promoter.

lncRNA

Example of a regulator not being a protein. They play a wide variety of regulatory roles, mostly in repression. Have to be transcribed to be active, sometimes in introns. Can act as cis or trans (only expressed regulator that acts in cis).

Histone reader-writer complex

Their DNA methylation is passed on during cell division. TFs could come and repress or turn on, and then others can come and make different changes.

Insulators and Barriers

Stop enhancers and silencers from acting on the wrong gene as they are far from the start site. Insulators divide DNA into looped regions. Insulators limit how far a cis element can interact from. Create loop of DNA and things can interact with the loop and only that loop. CTCF.

Eukaryotic networks

We can turn on transcription or something to regulate transcription.

Negative feedback loops (when we start transcribing it feeds back to turn the stuff off), positive feedback loop (turns on genes involved in same process).

Prokaryotic regulation

Multiple genes from a single promoter, gene expression depends on the environment. Operon, tryptophan repressor.

Operon

An operator. A secondary messenger informs of status. Each contains regulatory DNA sequences, which act as binding sites for regulatory proteins that promote or inhibit transcription.

Tryptophan repressor

Binds to operon in presence of T. When T operon is transcribe, everything comes together. Operon doesn’t want to waste energy so it binds to a repressor and turns off so RNA pol cannot be recruited.

Exonic splicing enhancer/suppressor

Control recruitment and how often something is included or excluded (ESE, ESS)

Negative control

Prevention of access to splice site by a repressor

Positive control

Where an exon is inefficiently read and left in and leads to recruitment of activator to splice sequence out. Removing sequence from final mRNA.

Leaky scanning of Kozak sequences

Can create versions of proteins that differ at N-terminus. Alternative splicing can create spliceoforms with different amino acid sequences. Post-transcriptional RNA editing can create versions of the protein with different amino acid sequences.

3’ UTR

On the end of 5’ promoter with N-terminus.

Proteins bind to cis-acting sequence often found in this area.

UTR

Control mRNA stability (capping A polyadenylation), translation, and localization.

Degradation through recruitment of endonuclease. Protein binding to block translation.

Block Shine-Dalgarno sequences in bacteria

It is temperature sensitive and increasing the temperature causes chain in hairpin, and pathogens change.

mRNA localization

Specific localization of RNA through multiple mechanisms. Sequences in the 5’/3’ UTR come into contact with things and they either anchor, protect or transport in local area. Useful when protein is in high demand in one area, one stays a stem cell and one differentiates.

P-bodies

Sites of mRNA degradation or storage of translationally repressed RNA. Abundant in decapping enzymes. Not membrane bound. Regulate whether RNA is localized or not.

Stress granules

Accumulates under stressful situations, works similar to P-bodies. Moves stuff into them until issues have passed.

Regulation using non-coding rna

lncRNA can bind complementary sequences and recruit proteins to act on those genes. They can recruit and have functions in regulation, can complementary base pair to target particular RNA or DNA.

X-inactivation

lncRNA Xist acts on chromosome and starts producing Xist RNA. When one starts to work, it is going to work to recruit mediators of epigenetic silencing.

RNA interference (RNAi)

More non-coding RNA. Superpower of complementary base pairing. A number of genes are regulated by this. Small pieces of RNA is made and it recognizes targets by complementary base-pairing and regulating a large number of eukaryotic genes.

Dicer enzyme

Cleaves dsRNA in the processing of miRNA

RISC complex

1 strand is degraded, leaving miRNA. Argonaute proteins in it are the key.

CRISPR

clustered regularly interspaced short palindromic repeats. Has viral and repeated sequences, helps with loading. Directs Cas if virus is reintroduced.

CRISPR/Cas9 Genome Editing

Swap out guide sequence and target double strand breaks. For permanent deletion, minimal change could still cause issues.

To avoid frameshift, an edited gene in place is added so nothing changes about regulation of transcription or leads to cancer.

crRNA

CRISPR used as a defense mechanism in bacteria using small noncoding RNA molecules (crRNAs) to seek out and destroy invading viral genomes through complementary base-pairing and targeted nuclease digestion.

CRISPR NHEJ

In mammalian cells, due to its efficiency and how many mammalian cells there are (less dangerous)

CRISPR HDR

In experimentation, may be chosen, but also may not due to its errors and off-target effects.