Cellular Biology - Exam Four

Mutation

misincorporation of nucleotides during DNA replication

Point Mutation

substitution of one base with another

Silent mutation

Doesn’t change the amino acid (shows the importance of codon redundancy)

Missense Mutation

exchanges one amino acid for another, changing the sequence of genes

Nonsense Mutation

produces an early stop codon; leads to a truncated protein

Frame shift mutation

addition or deletion of bases changes the reading frame of the triplet codon; all amino acids beyond the mutation are changed; frequently results in a premature stop codon

Nonsense mediated decay

mRNA transcripts that contain a premature stop codon are usually translated once and destroyed.

During splicing of the primary RNA transcript, the spliceosome does what?

leaves behind a complex of proteins known as the exon junction complex

When mRNA is translated the first time (during mRNA surveillance) the ribosomes do what?

Remove the exon-junction complex from the transcript

If there is a premature stop codon, the ribosome may what during mRNA survellience?

The ribosomes may dissociate before knocking off all of the exon-junction complexes

Any exon-junction complex not removed from the transcript will do what?

trigger the destruction of the mRNA by ribonucleases.

Micro RNAs (miRNAs)

RNA’s of 21~24 nucleotides in length that are encoded for by a genome and target a specific mRNA sequence whose translation needs to be prevented

Micro RNA’s are synthesized by what?

RNAPII

What happens during miRNA’s synthesis?

The primary transcript folds back on itself to form a double-stranded hairpin shaped pri-miRNA. The remaining single stranded sections of the pri-miRNA are trimmed within the nucleus by an enzyme called Drosha to produce a pre-miRNA. The pre-miRNA is exported out of the nucleus and the hairpin loop is cut out by a ribonuclease called Dicer (mature miRNA). The double stranded miRNA associates with an argonaute protein, which destroys one of the RNA strands. The single strand miRNA and the argonaute protein are incorporated into a protein complex called RISC. RISC allows the miRNA to form complimentary base pairs with the mRNA for which it is specific.

What happens if the base pairs in RISC are only partially complimentary?

The miRNA blocks translation of the mRNA into a protein

What happens if the base pairs in RISC are completely complimentary?

Argonaute cleaves the mRNA, leading to its degradation.

Where does protein synthesis begin?

On cytoplasmic ribosomes

Depending on the final destination of the protein being synthesized, where might the ribosomes and their peptide chains bind?

To the rough endoplasmic reticulum

What types of proteins are synthesized on cytoplasmic ribosomes?

those that are transported into the nucleus (ribosomal proteins) and those whose jobs are in the cytoplasm (glycolytic enzymes)

What types of proteins are synthesized at the rough endoplasmic reticulum?

proteins meant to be released from the cell(hormones) integral membrane proteins (cell surface receptors) and certain organelle proteins (lysosomal enzymes)

The primary structure of proteins contains?

all information needed for the protein to fold into its proper tertiary structure

If a protein is left in an isolated environment (like a test tube), the protein can?

fold into its proper tertiary structure without an input of energy

In the crowded environment of the cell, proteins may what?

become distracted and veer off from the correct folding pattern

Chaperonins do what?

provide a safe environment within the cell for the proteins to fold.

What are the two structures of the Chaperonin?

The GroEL and the GroES

What is the GroEL?

a structure that forms a chamber for protein folding

what is the GroES?

a structure that provides a lid for the chamber that GroEL creates

What is the Chaperonin’s mechanism?

When empty, the GroEL chamber is hydrophobic. The hydrophobic regions of misfolded/unfolded proteins will be drawn to the interior of the chamber.

What happens when a protein is bound to a Chaperonin?

The GroES structure will cap off the chamber and the chamber enlarges. The chamber will display hydrophilic residues and drops the bound protein into chamber. After 15 seconds, GroES removes itself from GroEL and the protein is released.

What happens when the chaperonin structures release the protein?

If it is properly folded, it can go off to do its job. If it isn’t properly folded, it can re-enter the chamber to try again.

What is the purpose of the nuclear Envelope?

It serves as a barrier between nucleoplasm and the cytoplasm

What is the sturcture of the Nuclear envelope?

Consists of a double membrane separated by an intermembrane space. The outer membrane is contiguous with the rough ER. The inner membrane is coated with a mesh work of intermediate filaments called the nuclear lamina.

What is the purpose of the nuclear lamina?

It provides mechanical support for the envelope and serves as a site for attachment of chromatin fibers.

The fusion of the inner and outer membrane of the nuclear envelope produce what?

Nuclear pores

What is the function of nuclear pores?

molecules move in and out of the nucleus through nuclear pores.

What regulates the movement of materials throughout the nuclear pore?

The nuclear pore complex

Describe the structure of the nuclear pore complex.

It has a cytoplasmic side, which contains cytoplasmic filaments, a nuclear side that contains a nuclear basket, and a central channel that is lined with nuceloporin proteins.

What is the purpose of the cytoplasmic ring?

Contains cytoplasmic filaments, which catch molecules tarted for entry into the nucleus

What is the purpose of the nuclear ring?

Contains the nuclear basket - which may play a role in regulating the exit of molecules out of the nucleus

What is the purpose of nuceloporin proteins?

the proteins contain clusters of phenylalanine-glycine repeats (FG). The FG motifs have a disordered structure that gives the proteins an extended flexible organization. They form a hydrophobic mesh work that blocks the free discussion of larger macromolecules through the complex

How are proteins targeted for entry into the nucleus?

via a nuclear localization signal. any protein whose final destination is not the cytoplasm has a signal sequence at its amino terminus. It is also rich in positively charged amino acids

What is the function of the amino terminus?

It is a short series of amino acids that serves as an address for delivery of the protein.

How are proteins moved into the nucleus?

the nuclear localization signal binds with the cytoplasmic receptor importin-a, forming a receptor-cargo complex. This complex then binds with importin-B which docks the protein at the nuclear pore by binding to the cytoplasmic filaments. The receptor-cargo complex is translocated through the pore. GTP-RAN binds to importin-B, dissociating the receptor-cargo complex and unloading of the cargo.

What happens after the “cargo” is unloaded at the nuclear pore?

GTP-RAN bound importin-B is recycled back to the cytoplasm through the NPC. The GTP is hydrolyzed to GDP, releasing importin B. Importin-A is transported back to the cytoplasm by a GTP-RAN exportin protein complex.

Proteins synthesized at the RER travel by what to Golgi complex?

membrane vesicles

What happens after proteins are brought to the golgi complex?

The proteins are modified and become “mature”; and then travel to their final destination via membrane vesicles

What is the structure of the RER?

network of flattened membranous sacs called cisternae, ribosomes bound to the cytosolic surface; inside the cisternae is the lumen/cisternal space. There are also protein channels within the RER membrane that connect with the cisternal space to the cytoplasm called translocons.

Cisternal Space structure and function?

The channel is shaped like an hourglass. In its inactive state the channel is plugged by a short alpha helix, and the channel has a side door that can open and close.

Describe the process of synthesizing secretory and organelle proteins.

mRNA binds to a cytoplasmic ribosome. The amino terminus of the nascent peptide chain contains a signal sequence that targets it towards the RER (made of 6-15 hydrophobic acids). As the peptide chain emerges from the ribosome, the signal sequence is recognized by a signal recognition particle (SRP). The SRP binds to the signal sequence and the ribosome, temporarily halting protein synthesis. The SRP then binds to its receptor on the surface of the RER, and the ribosome binds to a translocon. The SRP releases the signal sequence, inserting it into the translocon channel. The contact of the signal sequence with the interior of the translocon leads to the displacement of the alpha helical plug and opening of the passageway. Translation resumes and the new peptide chain is threaded through the translocon into the lumen of the RER.

Describe the synthesis of integral membrane proteins that span the membrane ONCE.

As the peptide chains is being synthesized and threaded through the translocon, the door of the translocon continuously opens and closed. If the section of the protein in the channel as the door opens is hydrophilic, it stays in the channel to continue its journey. If it’s hydrophobic, it will move out into the membrane. Most transmembrane segments have a positively charged amino acid flanking the end that is facing cytoplasm, which determines their orientation in the membrane. The transloon can flip the transmembrane domain to place it in its proper orientation.

Describe the synthesis of integral membrane proteins that span the membrane multiple times

the orientation of the segments is determined by the direction in which the first transmembrane domain is inserted. All subsequent transmembrane segments are going to weave in and out of the membrane in opposite directions. All integral membrane proteins remain embedded in the membrane from this point on.

Describe the processing of the protein within the RER lumen.

The enzyme signal peptides removes the signal sequence. The chaperones help the protein fold properly. Protein disulfide isomerase (PDI) catalyzes the formation of disulfide bonds between cysteine amino acids, which helps maintain the proper tertiary structure. The addition of sugar chains creates a glycoprotein, where the added sugars play a critical role in structure, function, and the localization of the protein. The sugars can be N-linked, or attached to the amino group of the AA asparagine. Or O-linked, or attached to the hydroxyl group of the AA serine or threonine.

Describe the basics of N-linked glycosylation

The same basic sugar core chain is added to proteins within the RER and the sugar core is modified within the Golgi.

Describe the synthesis of the sugar core during N-linked Glycosylation.

Built on the dolichol phosphate, a lipid located in the RER membrane. The first 7 sugars are added to the lipid on the cytosolic side of the membrane (two N-acetylglucosamines and five mannose sugars). Then the dolichol flips across the membrane and the remainder of the sugars are added on the luminal side of the RER membrane (4 mannoses and 3 glucoses). The fully assembled oligosaccharide chain is then transferred to an asparagine on the nascent peptide chain as it is being translocated into the ER lumen. Two of the three glucoses are removed.

Describe Quality control of the proteins

Before proteins exit the RER they are checked for proper folding. The chaperone Calnexin binds the new peptide chain via the single glucose remaining on the core sugar and the protein is released. A monitoring enzyme UGGT, checks the protein for proper folding. Improperly folded proteins have exposed hydrophobic regions. If folded properly, protein is transported to the golgi. If improperly folded, UGGT adds a glucose back on the the core sugar. Calnexin rebinds the protein and gives it another change to fold.

Describe Protein Destruction

if the protein never folds properly is is sent back out into the cytoplasm for destruction by the proteasome, which is a protein degrading machine that destroys misfolded or old proteins.

What is the structure of a proteasome?

A cylinder composed of four rings stacked on top of one another. The two outer rings are composed of alpha subunits and are covered with caps. The two inner rings are composed of Beta subunits. These are proteolytic enzymes whose active sites face the inside of the cylinder, who cut the proteins int o small pieces.

What is the proteasome mechanism?