Molec. Cell Exam 3

Characteristics that make RNA polymerase unique in prokaryotes

-10 and -35 promoters and the sigma subunit

How is transcription initiated in prokaryotes?

By the release of the sigma subunit

How is transcription terminated in prokaryotes?

A G-C rich sequence followed by a run of A’s is reached and a stem loop is formed

Polymerase 1

Found in eukaryotes for the formation of rRNA. The building block subunits are 5S, 5.8S, 18S, and 28S

Polymerase 2

Found in eukaryotes for the formation of mRNA, miRNA, lncRNA, and snRNA

Polymerase 3

Found in eukaryotes for the formation of tRNA and scRNA

Ribose methylation and uridine isomerization

Post-transcriptional modifications of rRNA

RNase P that cleaves extra sections of RNA

Post-transcriptional modifications of tRNA

Inosine that helps to stabilize the structure

A modified base created in the post-transcriptional modification of tRNA

Splicing introns, 5 prime cap, and poly-A tail

Post-transcriptional modifications of mRNA

M7G- m7 methyl guanosine

Another name for the 5 prime cap

Spliceosome

This complex is composed of small, nuclear ribonucleic proteins

U2, U5, and U6

These are the snRNPs (snRNAs) that will cleave the 5 prime site, form the lariat, cleave the 3 prime site, and ligate the exons

Group 1 self-splicing introns

Do not form a lariat, they just cleave the 5 prime site and rely on a guanosine co-factor. The cleavage at the 3 prime site is spontaneous due to exon 2 attacking exon 1 and the exons are ligated.

Group 2 self-splicing introns

Have adenosine in the intron that cleaves the 5 prime site, leading to the formation of a lariat. The cleavage at the 3 prime site is spontaneous due to exon 2 attacking exon 1 and the exons are ligated. 

DMD gene

Produces dystrophin, which stabilizes actin by anchoring it to the plasma membrane. There is a splicing error that occurs where exons 48-50 are spliced out, which changes the splicing sequences. This error leads to a frameshift mutation when exon 47 binds to exon 51 and creates a pre-mature stop codon, which results in a nonfunctional protein. There is a drug that blocks exon 51 so that it is internally deleted, but there is still a partially functioning protein. 

Lac operon

It is polycistronic (multiple genes on a promoter) and found in prokaryotes that metabolizes and ferments lactose. On this, there is a promoter and operator (which will determine if the promoter will be used). This is only turned on when glucose is low and lactose is present.

beta-galactosidase, lactose permease, and transacetylase.

What are the three genes that metabolize and ferment lactose?

Negative control of lac operon

The I gene that codes for a repressor protein. The repressor will bind to the operator and block the RNA polymerase complex. The repressor can be inactivated by allolactose, which is a byproduct of lactose metabolization.  

Positive control of lac operon

Adenyl cyclase that converts ATP to cyclic AMP. Adenyl cyclase is a major secondary signal that binds to the CAP protein. The CAP protein binds upstream to the promoter and is a transcription factor that recruits RNA polymerase to the promoter. Adenyl cyclase can be inhibited by alpha-ketoglutarate that is used in the Krebs cycle (produces high glucose concentrations). The presence of glucose will switch the operon off and there will be no transcription.

Eukaryotic promoters

The TATAA box is the main factor, but these can also be variable. With these, there are also enhancers and mediators. Enhancers (SB40) work to enhance transcription regardless of their position relative to the promoter. Enhancers will work on DNA loops (condensed DNA) by attracting cohesin and bringing the transcription factor and promoter together. Mediators work with transcription factor 2 and polymerase 2 to stabilize the complex.

Electrophoretic-mobility assay

It is a process that finds (identifies) transcription factors. DNA sequences with promoters are put on DNA gel, incubated with protein, and/or are incubated with the protein and antibody. Each method provides a different result (mobility shift) which indicates that the protein or the protein and antibody are binding to DNA and could be transcription factors.  

Chromatin immunoprecipitation

The cells are treated with formaldehyde, which is a crosslinker. The transcription factor or protein associated with DNA will stay stuck to the cell. The cells are lysed and then are incubated with antibody against the protein in homogenate. Sepharose protein A (neutralizes antibodies) binds, and then the compound can be purified using centrifugation or a column. Finally, the cells can be chipped (heated to 65-95 degrees Celsius), which causes formaldehyde to release, and the DNA can be sequenced to determine protein association. 

DNA-affinity chromatography

There is an agarose bead and GC box DNA that goes through a GC box DNA column. Then, a mix of cells proteins is run through the column, and the proteins will flow through. The column consists of a high salt buffer solution. 

Serum response factors

These are factors that are associated with mitogens (chemicals that cause cell division and differentiation). They are transcription factors that have CC bases on the 5 prime end, A and T bases in the middle that are variable, and GG bases on the 3 prime end.  

Transcriptional activators

Proteins that bind to DNA and stabilize mediator/initiation complexes. They modify chromatin structures in eukaryotes. 

Eukaryotic repressors

They compete with activators for the binding site. They can bind to DNA and mediator and block RNA polymerase 2 (activation of initiation complex), which blocks transcription

Histone acetylation

Modifies lysine residues with acetyl groups

Histone modification

Can be methylated and phosphorylated. DNA with promoters have a pattern of H3K4me3 (histone 3, 4th lysine in the sequence, and tri-methylated). DNA with enhancers have a pattern of H3K4me1 (histone 3, 4th lysine in the sequence, and mono-methylated). H3K27ac is histone 3, 27th lysine in the sequence, acetylated. 

Chromatin remodeling factors

These can be used to: 1) slide histone octamer along DNA, 2) change the shape of nucleosomes, and 3) dissociate histones from DNA. Elongation factors bind to phosphoserines that contain histone acetylases and CRFs that remodel chromatin upstream of polymerase 2, so DNA is accessible to transcribe. The nucleosome is modified with histones, are then distributed to progeny strands, new nucleosomes get incorporated, and then parental modified histones direct modification of new nucleosomes.

Polycomb Repressive Complex 2

It is like methyl transferase. It copies tri-methyl groups onto new nucleosomes. Stress can cause methylation, limiting the DNA available for transcription. Changed cells are passed into new cells or gametes (inheritance of modified nucleosomes). Linker DNA can also be methylated (cytosine and guanine sequences) and passed to offspring (genetic imprinting). The methylation can be reversed by TET that utilizes base excision repair.

aminoacyl tRNA

Fully charged tRNA that has an anti-codon on one end and an amino acid on the other end.

Ribosomes

Composed of proteins and rRNA molecules

A site

This site on ribosomes is used to accept tRNAs with amino acids attached

P site

This site on ribosomes is used to polymerize amino acids

E site

This is the site on ribosomes where the tRNAs exit without the amino acid

Prokaryotic

These cells have 70S ribosomes that are broken down into a larger unit and smaller unit. The larger unit is 50S ribosome size and is made up of 23S and 5S rRNAs. The smaller subunit is 30S ribosome size and is made up of 16S rRNAs

Eukaryotic

These cells have 80S ribosomes and are broken down into a larger unit and smaller unit. The larger unit is 60S ribosome size and is made up of 28S, 5.8S, and 5S rRNAs. The smaller unit is 40S ribosome size and is made up of 18S rRNAs. 

Prokaryotic mRNA

There are no end modifications (transcription and translation are coupled), there are upstream and downstream untranslated regions that can play a role in regulation, there are multiple stop and start codons, and they are polycistronic meaning they can create multiple proteins. This allows them to be able to respond to the environment more quickly. 

Eukaryotic mRNA

There are post-transcriptional modifications, there are upstream and downstream untranslated regions that can play a role in regulation, there is a single start and stop codon, and they can only create one protein after splicing (introns and exons). 

Shine-Dalgarno box

This is found only in prokaryotes and is a unique sequence that is upstream from the start codon and has complementary base pairing to the ribosome (16S rRNA) on the 3 prime end. This allows the cell to be able to distinguish between initiator AUG sequences and internal AUG sequences. 

Polysomes

They are multiple ribosomes on a strand of mRNA. Almost like "beads on a chain."

EIF 1 and 3

These will bind to the small subunit of ribosome during translation initiation

EIF 2

Binds to the tRNA (which should have a methionine as the first amino acid) during translation initiation

EIF 4

Forms a complex that binds to mRNA during translation initiation. Once the mRNA is bonded, the complex undergoes scanning which consumes an ATP, but tRNA will be lined up with the start codon and complementary base pairing will begin

EIF 5

Will bind to GTP and lead to the assembly of the large subunit with the small during translation initiation. The EIFs will get displaced, and the A site in the ribosome will be open.

Internal ribosome entry sites

This is a variation in translation initiation. These allow translation to be initiated when the cell is stressed (low ATP or GTP). 

Elongation during translation

Conducted by eEFs. EEF-1 alpha recruits the next tRNA (every tRNA that is added consumes GTP). EEF2 provides GTP for translocation. This occurs simultaneously with the formation of the peptide bond (where one tRNA has two amino acids on it and the tRNA in the P site goes to the A site). 

Translocation

When the ribosome slides down the mRNA toward the 3 prime end (one codon) while the tRNAs stay in place. Essentially, the tRNA in the A site will move to the P site, the tRNA in the P site will move to the E site, and the A site will be open to accept another tRNA. 

Translation termination

The A site will be positioned over a stop codon, which recruits release factors (RFs). The RFs cause dissociation of polypeptide off the last tRNA

Regulation of ferritin

Involved in the transport and utilization of iron. When there is ample iron, this binds to the IRP (iron regulatory protein) and deactivates it (degradation). When there is scarce iron, it binds to the IRP that stays present and then binds to the mRNA at the IRE (iron regulatory element) and blocks the initiation complex (EIF-4).  

Translation repressors

Blocks EIF-4 from making the loop with the 3 prime untranslated regions

miRNAs

Short sequences complementary to RNAs. These create a loop that is affected by dicer and RISC that creates a RISC complex that will be cut into single strands (deadenylating). The RNA will be blocked from translation and polyadenylated (chews up poly-A tail faster).  

Phosphorylated

During stress or growth factor starvation. EIF 2 can be which stops the GTP-GDP exchange.

Chaperones

Are used to assist in folding. They bind to the polypeptide and function as enzymes. Are favored in warmer environments because heat shock drives the expression of heat shock proteins

Chaperonin

When the proteins are in a partially folded intermediate, they are transferred to a complex with a barrel shape that creates microenvironments that support protein folding. GROel acts as the barrel, and GROes acts as the lid. 

Amyloid formation

A native protein that is mostly alpha helix based will undergo a misfolding event (shift in tertiary structure) and enters a state that is primarily beta-sheets where more misfolded proteins can stack. 

Prion propagation

An amyloid disease caused by misfolding. The addition of one of these leads to elongation, and as more are added, the protein will fragment. Humans can experience this if they consume food that is contaminated (transmissible spongiform encephalopathies (TSE)). 

Protein disulfide isomerase

Works with disulfide bonds. It will donate bonds to the cystines in the folded protein and accept hydrogen bonds.

Peptidyl prolyl isomerase

Works with proline, which creates two covalent linkages (and turns the chain). When the R group and the proline are on the same side, it is in the cis position. PPI will convert it to the trans position, when the proline and R group are on opposite sides.