Genetics Exam 2

Explain the experimental results that led to the "one gene one enzyme" hypothesis

The "one gene one enzyme" hypothesis was formulated based on experiments conducted by George Beadle and Edward Tatum in the 1940s. They used the fungus Neurospora crassa to demonstrate that specific genes are responsible for the production of specific enzymes, as mutations in these genes resulted in the inability to synthesize certain nutrients.

Their work provided the first direct evidence linking genes with specific enzymatic reactions, highlighting the role of genes in determining the biochemical phenotype of an organism.

Explain the complementation test

Test to determine whether two mutations that produce similar phenotypes are in the same or different genes

What are complementation groups and how are mutants assigned to complementation groups?

Complementation groups are sets of mutations that fail to complement each other, indicating they are in the same gene. Mutants are assigned to these groups based on their inability to produce a wild-type phenotype when crossed with other mutants. If two mutants are in different genes, their combination will produce a wild-type phenotype.

What is minimal media versus complete media? Why were the two media conditions used in Beadle and Tatum's groundbreaking experiments?

Minimal media contains only the essential nutrients required for growth, while complete media contains all nutrients including vitamins and amino acids. Beadle and Tatum used both these media conditions to determine the specific nutritional requirements of mutants, demonstrating the relationship between genes and metabolic pathways.

What is the central dogma of molecular biology?

The central dogma of molecular biology describes the flow of genetic information from DNA to RNA to protein. It outlines the processes of transcription and translation, emphasizing how genes are expressed to produce functional proteins.

What are the molecular differences between RNA and DNA?

RNA contains ribose sugar, while DNA contains deoxyribose. Additionally, RNA uses uracil in place of thymine, and RNA is typically single-stranded, whereas DNA is double-stranded.

Define the different elements comprising a eukaryotic "gene body" as defined in class. How is this different from prokaryotes?

A eukaryotic "gene body" consists of exons, which are coding sequences, and introns, which are non-coding sequences that are spliced out during RNA processing. In contrast, prokaryotic genes typically lack introns and are organized as continuous coding sequences without the need for RNA splicing.

Does a gene consist of more than its gene body?

Yes, a gene also includes regulatory elements such as promoters and enhancers that control its expression.

Compare exons and introns. Which have coding sequence and which have untranslated regions? Which appear in mature mRNAs?

Exons are the coding sequences that remain in mature mRNAs after splicing, while introns are the non-coding sequences that are removed during RNA processing. Exons contain the information for protein synthesis, whereas introns are typically found in the untranslated regions.

What is a cis-regulatory element? Can these elements always be identified computationally? Where do they reside in DNA?

A cis-regulatory elements (enhancers) are a region of non-coding DNA that regulate the transcription of nearby genes. These elements can be identified computationally, but not always, as their function may depend on context and specific interactions with transcription factors. They typically reside upstream or downstream of the gene they regulate. They can be activators or repressors that are found on the same strand of DNA.

Define chromatin and its composition. What are some differences between heterochromatin and euchromatin?

Chromatin is a complex of DNA and proteins, primarily histones, that package DNA into a compact structure in the nucleus. Heterochromatin is densely packed and transcriptionally inactive, while euchromatin is less condensed and actively involved in transcription.

What are the components and functions of nucleosomes?

Nucleosomes are the fundamental units of chromatin, consisting of a segment of DNA wrapped around a core of histone proteins. They play a crucial role in packaging DNA into a compact structure and regulating gene expression by controlling access to the DNA.

Histone modifications are essential for regulating chromatin state. What are major types of modification and the enzymes that participate in them? What are the consequences for transcription?

Histone modifications include acetylation, methylation, phosphorylation, and ubiquitination, which are catalyzed by specific enzymes such as histone acetyltransferases (HATs), histone methyltransferases (HMTs), and kinases.

Methalyzation - Histone methylation is defined as the transfer of one, two, or three methyl groups to lysine or arginine residues of histone proteins by histone

-methyltransferases (HMTs) and Protein Arginine methyltransferase (PRMT)

-Can either increase or decrease transcription rates by condensing/ uncondensing

-DNA methylation decreases transcription

*Acetylation - The modifying enzymes involved in histone acetylation are called histone acetyltransferases (HATs)

-Histone deacetylase (HDAC) removes acetyl groups

-Increases transcription rates by uncondensing

Distinguish ATP-dependent chromatin remodelers (e.g., SWI/SNF or chromodomain factors) from histone modifying enzymes (e.g., HDACs, HMTs).

-The histone remodelers can slide, displace and modify nucleosomes in order to free up DNA for transcription or cis regulatory/promoters for aiding in transcription

-Histone modifying enzymes can not do as much as the remodelers mainly they phosphorylate, methylate, and acetyalte histones to alter tightness of DNA wrapping to facilitate or reduce trancription,

Describe the various ways that ATP-dependent chromatin remodelers can affect DNA binding site accessibility

Repositioning nucleosomes

Ejecting histones from nucleosome

Unwrapping DNA from around histone core

Exchanging or Ejecting Dimers (Dimer is 2 histones)

What are the Pre-initiation and Mediator Complexes and how do they promote transcription? How do the proximal promoter and “distal” cis-regulatory elements (occupied or unoccupied) fit into the process?

Pre initiation Complex - a complex of 100 proteins that is necessary for the transcription of protein-coding genes in eukaryotes and archaea.The preinitiation complex positions RNA polymerase II at gene transcription start sites, denatures the DNA, and positions the DNA in the RNA polymerase II active site for transcription

Mediator Complex- Promote transcription by allowing transcription factors bound cis regulatory elements far upstream or downstream to become involved acting as activators or potentially repressor elements. Helps Pol II bind and Helps get PIC to bind

Proximal Promoter and cis regulatory elements proximal promoter bind proteins that facilitate the process of transcription (about 250 bps upstream) without them the Pre-initiation Complex and mediator are not held in place properly and cis regulatory elements are important for catalyzing large amounts of transcription rather than just low amounts (work on a higher level to increase/decrease not yes/no); Cis - On the same strand of DNA ; Trans - somewhere not on the same DNA strand

How do “basal” or “general” transcription factors compare with tissue- or cell-type specific transcription factors?

Basal/general transcription factors are found in most or all locations where transcription is occurring. Cell/tissue specific transcription factors are unique to a specific cell or tissue transcription

Several DNA motifs in promoters are essential for the commencement of transcription from particular genes. What are two of these motifs and how do they function to promote transcription?

-TATA box - a DNA sequence about 30 bps upsteam that binds TATA box protein (5'-TATAAA-3) within the core promoter region where general transcription factor proteins and histones can bind.

-Initiator - always found -2 to +4 helps with start of transcription

-BRE - Helps bind proteins to TATA region

-DPE - Downstream Promotor

-Motif - matching pattern associated with something

-transcription start site (TSS)

Describe the possible steps by which a gene in a region of closed chromatin begins to be transcribed

1. Pioneer factors show up and open/stabilize the heterochromatin

*Also ATP dependent chromatin remodelers and histone modifying enzymes may be present to help open up the chromatin

2. Other proteins and transcription factors start to bind

3. The Pre-Initiation Complex assembles and recognizes promoter motifs that act cooperatively to promote transcription

4. Mediator assembles and pull cis regulatory elements with attached TFs to the site of transcription

5. RNA Pol II binds and starts to transcribe

What are some general ways in which transcription factor activity can be regulated (for transcription factors acting as repressors or activators).

Ligand binding, protein-protein interactions, post-translational modifications (like phosphorylation and acetylation), subcellular localization, protein stability, and chromatin remodeling

What is the 5' cap on mRNA, how is it synthesized and what does it do

A modified guanine nucleotide added to the very first position of a messenger RNA molecule during transcription, acting as a protective cap that helps the mRNA efficiently bind to ribosomes for translation and protects it from degradation by exonucleases

What strand of DNA is transcribed by RNA polymerase II and in what direction? In what direction is the RNA molecule synthesized?

The template strand is the strand of DNA that gets transcribed by RNA polymerase II in the 3’ to 5’ direction. RNA molecule is synthesized from the 5’ to 3’.

What is RNA Pol II pausing and how is it regulated? Speculate on why pausing might occur

- RNA pol 2 has early elongation called pioneer escape for 20-60 nucleotides and then undergoes pausing of pol 2

-by stopping this is another check to see if the gene is going to be transcribed

- pausing depends on interactions with the: mediator complex, NELF, DSIF, promoter elements, and stable RNA/DNA hybrids which might be hard to break (ex C-G 3 h bonds)

- proteins and elements of the promoter itself are involve in the pausing

- productive elongation then continues rapidly through termination

- pausing may keep genes POISED for expression and allow additional regulation and signal integration while maintaining access to the PIC

What are the basic events of mRNA splicing and how do specific sequence regions delineate where these events happen? How specific and predictable are these sequences?

- mRNA splicing typically occurs at the GU-AG splice sites with interrupting branch sites

- splice donor before a GT and splice acceptor after an AG typically, branch site is in the middle about 30 nucleotides long ad is usually CT and ACT

- sites are functionally equivalent across introns so correct splicing depends on the simultaneous recognition of corresponding GU-AG regions, if misses one AG or GT then could accidentally cut out Exon etc.

-spliceosome: small nuclear ribonucleoproteins (snRNPs) each comprise a small RNA with about 20 proteins

-four spliceosomes work together and this is done while RNA is still being transcribed

- cut at splice donor site and then branching occurs to the branch site and a 2' to 5' bond is made, creating a loop called a lariat

-then a cut is made at the splice acceptor and the two exons are put back together

- there is lots of room for error in splicing and misplacing can lead to disease

- may different variability and alternative splicing produces many different kinds of regulation of genens

- 20,000 protein coding genes to over 100,000 protein coding mRNAs

-variable features at core promoters and TSS (may have multiple promoters and which ones used determines which RNA is made), transcriptional termination or polyadenylation, also varies in splicing to retain diff exons ad introns

- alt splicing may retain (or not) certain exons or introns

-95-100 percent of mRNAs w more than one exon yield multiple mRNAs

How common is alternative splicing and what are some examples of the phenomenon? What is an overall consequence of alternative splicing (e.g., in the context of the "one-gene / one enzyme" hypothesis)?

alternative splicing is persuasive

- 95-100% of pre-mRNAS with more than one exon yield multiple mRNAS (2 to thousands of variants per gene)

- splice acceptor and donor sites are invariant (how does cell know where to do alt splicing?)

- consequence of alt splicing is many mRNAs and proteins made from one pre-mRNA due to alternative splicing, one gene one enzyme hypothesis false

- we only know general factors affecting (cis and trans splicing regulators, transcriptional rate due to histone mods) but we do not know EXACTLY how splicing occurs

Explain the roles for splice site enhancer and suppressor sequences and protein in determining whether splicing occurs at a particular site

- cis regulators are the RNA motifs and are recognized by proteins that will determine whether expressed or not (ex. exonic/intronic splicing enhancers/suppressors, ESE)

- trans regulators are proteins that bind the RNA cis motifs: serine arginine rich proteins promote splice site usage and compete with repressive heterogeneous nuclear ribonucleoproteins (hnRNPs) and tissue specific regulators (these tend to repress splicing sites)

- this occurs AT THE LEVEL OF RNA, only recognize RNA after transcribed, diff combos of factors at different times leads to different splice forms coming out

- possible for introns to be retained Premature stop codon causes whole thing to be targeted for degradation

What is the origin of the poly(A) tail found on mature mRNA? Why is having a poly(A) tail

important?

-pre-mRNAS are polyadenylated at 3' end when transcription terminates

- RNA binding proteins associate with Pol 11 and recognize polyadenylation signals (AAUAA) in the 3' UTR, several present and choice is regulated

-RNA pol 11 transcribes the sequence and is recognized by factors including Poly A polymerase

-endonuclease binds and cuts at the G/U- rich target site 11-30 nt beyond the poly a signal

-poly a polymerase adds a poly A tail of 200-250 ATPs

-Functions poly A tail: binding sites for Poly (A) are RNA binding proteins in the nucleus (PABPN) and in the cytosol (PABPC)

PABPN: promotes Poly A polymerase activity (adds more A nucleotides), interacts w cleavage and polyadenylation factor to influence Poly A length and cut transcript, creates circular structure, functions in RNA export to cytosol

PABPC: interacts w eukaryotic initiation factors needed for ribosome function (elF4G, E), maintains loop structure critical bc protects from being chewed up by endonuclease (cannot access ends of RNA), promotes ribosome release (eRF3,1), blocks degradation of mRNA at 3' and 5' ends

-shorter poly A tail may limit opportunity for PABPC binding, translational efficiency and protection from nucleus

What explains the difference between the numbers of codons and the types of tRNA found in eukaryotic cells?

- at least one tRNA for each AA but not necessarily all 61 corresonding to each sense codon

-due to wobble position this accommodates degeneracy w promiscuous pairing of standard and modified bases

- given Phe tRNA can recognize more than one Phe codon

- modified nucleotides can recognize other, example anticodon I can recognize codon U, C, A

-dont need 61 tRNA genes to recognize all 61 possible codons

What are the events of translation initiation, elongation, and termination?

- initiation: mRNA has to associate with PABPC and elFs, small subunit recognizes the 5' methylated RNA cap and attaches to the 5' UTR, scans until it gets to the start codon (AUG) and then can accept the MET tRNA, small subunit charged with MET tRNA causes large subunit to come in and makes full structure, most proteins first Aa (AUG) sequence matters but also sequence around it called Kozak sequence (usually 3 U’s are AG, Us usually CG, and DS usually G) these are favorable for small ribosomes and without Kozak sequence not translated as efficiently, adds another level of control, elFs helps 40s join to 60s

- elongation: tRNA accepted into A site, links two AA detaching from the tRNA and moves into E site, one from A site moves to P site and E site leaves

- termination: release factor enters A site when hits the termination codon, causes ribosome to get off of the mRNA

What factors besides ribosomes are necessary for translation initiation?

1. 40s ribosome subunit must associate with eukaryotic initiation factors and MET tRNA, mRNA associated with PABPC and elFs

2. mRNA and 40s ribosome associate together

3. 40s subunit scans to get AUG start codon

4. elFs helps 60s join 40s

5 mature 80s starts translation

Many (but not most) genes have mRNAs with upstream open reading frames in their 5' UTR. What is a commonly accepted function of these upstream ORFs and what observations support this idea? What kinds of genes seem to have these upstream ORFs?

-uORFs reduce translational output for mRNAs that encode very potent proteins (growth factors), this reduction inn protein is normal and important for regulating differentiation in some contexts (nervous system)

-evidence: 5'UTR of PTCH1 hooked to fluorescent reporter gene, when 3 US ORFs were removed 5 fold more fluorescent protein was made, when 1 uORF mutated doubling in fluorescence intensity

- shows that uORFs in 5' UTR put the breaks on translation, makes translation LESS efficient

-often have uORFs for genes whose protein products are very potent (easy for too much protein to be made which is deleterious)

-with neuron cells: WT differentiate correctly into Foxa2, take one uORF that's missing and use PTCH coding sequence so much more PTCH protein made leads to much less differentiation to the FOXa2 state, too much PTCH1 poisons cells and doesn't let them differentiate

-why uORFs lower translational efficiency? multiple AUG means multiple places ribosome can bind and if there prevents other ribosomes from binding at the same time, where it recognizes the first AUG has a huge impact, multiple opportunities to start on small uORF and detach before moving protein, small majority would miss the stop and actually translate, evolutionary favorable to make small peptides than too much of a protein, uORF dont have good Kozak sequences recognized in some cases but also not in other cases

What are miRNAs and where are they located in the genome? How do miRNAs contribute to regulating transcript and protein abundance?

-a class of functional RNA that regulates the amount of protein produced by a eukaryotic gene

-microRNAS

- key regulators of mRNA abundance

-1900 miRNA genes in human genome each multiple target mRNAs

-forms miRISC can target mRNAs for degradation or block translation

mRNA abundance is relatively easy to measure whereas measuring protein abundance can be more difficult. What are the limitations of inferring protein abundance from mRNA abundance? and vise versa

-gene expression and activites of gene products are regulated at many levels: abundance at one level does not often predict abundance or activity at another level

- Many steps where can have regulation from gene to protein: genome (whether gene expressed), transcriptome (bursts transcription, splicing, transport, volume control how long transcripts persist due to Poly A and miRNA), translation (how efficient and how long), proteome (folding and PTM)

- risks bc mRNA may be changed in various ways from its initial point as a gene due to splicing, Bursts transcription, Poly A, miRNAs etc.)

- predicting abundance of a protein based on abundance of mRNA is NOT easy (NOT 1 to 1), many places in between where mods can occur, mRNA is a poor predictor of protein content