Describe the RER and Ribosomes.
- The RER has a rough appearance when viewed with electron miscroscopes, this is becasue it is studded with complexes called ribosomes.
- Ribosomes are a type of enzyme made up of rRNA. They are composed of 2 subunits and are the major enzyme responsible for translation, the final step in protein synthesis in the Central Dogma.
- Ribosomes assemble at the RER when a protein that must go to another organelle needs to be produced. This is called the endomembrane system.
- Ribosomes can also be found freely floating in the cytosol. These ribosomes only produce proteins that stay in the cytosol.
Describe the SER.
The smooth ER does not have ribosomes on it, giving it a smooth appearance when viewed with an electron microscope. It has the following functions:
- Synthesis of lipds, phospholipids and steroids
- Location where glucose-6-phophate can be converted to glucose
- In muscle cells, the SER regulates calcium ion concentration, which is important for muscle contraction
Briefly describe the components of the ER.
- The Nucleus: connected to the ER cristerna by the outer layer of the nuclear envelope
- Nuclear Pore: some of the nuclear pores connect the nuclear envelope with the ER membranes so molecules can pass through these structurs freely
- RER: dotted with ibosomes, proteins involved in the production of other proteins - the RER is the site for protein translation and some modification
- SER: does not hae ribosomes on it, and is responsible for lipid processing
- Ribosomes on the RER: responsible for protein production and translation
- Proteins to be Transported: proteins have been made in the ER shuttle to the Golgi for further modification
- Transport Vesicle: these are used to carry proteins that still need to be m odified between the ER and the Golgi apparatus
- Golgi Apparatus: this organelle is involved in protein modification and transport
- CIS face of the Glgi Apparatus: side of the Golgi apparatus that faces the ER
- Trans face of the Golgi apparatus: faces away from the ER
- Cisternae of the Golgi Apparatus: these are flattened disks of membrane - the golgi has different enzymes and functions in the different cristernae
What are the types of RNA?
Messenger RNA(mRNA): RNA that will be translated into protein - unlike rRNA and tRNA, it is a coding template for peptides - Any RNA molecules that do not serve as a template for protein are called noncoding
Ribosomal RNA (rRNA): make up the ribosome complex to translate messenger RNA into proteins - There are two subunits that make up a ribosome that are different sizes
Transfer RNA (tRNA): serve as a linker molecules that link specific building blocks of proteins to the mRNA that is cding the peptide during translation
Where does transcription occur?
In the nucleus.
Describe RNA polymerase.
The actual synthess of RNA from DNA is facilitated by an enzyme, termed RNA polymerase. Eukaryotes have 3 types, each of which makes a different kinf of RNA. RNA polymerase II synthesizes mRNA, which is the polymerase we will focus on. RNA polymerase I is responsible for catclyzing most of the rRNA required for a functional ribosome. RNA polymerase III synthesizes transfer RNA, as well as some other RNA molecules.
Describe transcription factors.
Not all genes are transcribed at all times within the cell. Certain proteins bind to regulatory areas of the gene, signalling to the transcriptional machinery which genes need to be transcribed. These proteins are called transcription factors. A transcription factor is a protein that binds to specific DNA sequences, and buu doing so controls the rate of transcriptioin from DNA to messenger RNA. Transcription factors perform this function alone or with other proteins in a complex. Transcription factors can promote or block the transcription of genes by altering the ability of RNA polymerase to start transcription.
Outline the stages of transcription.
- Initiation of RNA Transcription
- It begins with the binding of transcription factors to the transcription start point
- Upstream of the start point is a region of the gene termed the promoter
- The promoter region closest to the transcription start point is the core promoter, since it is essential for transcription
- Transcription factors bind to the core promoter at a sequence called the TATA box
- Once the transcription factors are bound to the TATA box - RNA polymerase II can bind to the promoter
- Given that transcription depends on the binding of a transcription factor to this core promoter, somthing that interfered with this process, like a mutation in this sequence, could silence the entire gene
- Binding of all these transcription factors facilitates transcriptioin by 3 mechanisms:
- -> Guiding RNA polymerase II to the correct DNA strand
- -> Unwinding the double stranded DNA enough for RNA polymerase II to access the gene being transcribed
- -> Activiting the enzyme function of RNA polymerase II by adding two phosphate groups to it (a process called phosphorylation) - Elongation of RNA Transcript
- The moving of the transcription complex (made up of RNA polymerase and the transcription factors) forward is the second stage
- RNA polymerase extends the RNA molecule 5’ to 3’ while reading the DNA template strand
- The entire region of unwound DNA, called the transcription bubble, is covered by the RNA polymerase - this protects the unwound single-stranded DNA from damage - Termination of RNA transcription
- The termination step is the least understood in eukaryotes, since eukaryotes do not have defined methods to step transcription
- Research has indicated that there are proteins that can bind to RNA polymerase II to indue termination
- Transcription normally ends when the RNA is literally cut from the RNA polymerase by a separate enzyme
- Unlike DNA polymerase, RNA polymerase does not have any proofreading function, and has a much lower fidelity - to compensate for this, it will make several short RNA molecules until one has the proper complimentary hydrogen bonds with the DNA template strand; this ensures correct mRNA molecule is made
Overview
- Transcription factors bind to the TATA box in the promoter, and guides RNA polymerase to bind to the promoter
- DNA is unwound at the promoter, exposing the template strand
- RNA polymerase synthesizes the first few nucleotides of an RNA molecule while remaining stationary at the promoter
- RNA polymerase moves along the transcription bubble, synthesizing the remainder of the RNA molecule - elongation
- RNA polymerase reaches a location on the template strand where no additional ribonucleotides are added to the RNA
- The transcription bubble collapses, the RNA molecule dissociates from the template, and the RNA polymerase falls off the DNA
What are the similarites and differences between replication vs. transcription?
Where it occurs:
R: In the Nucleus
T: In the Nucleus
What it produces:
R: A replication of a cells entire DNA
T: Production of RNA that are made from one or a few genes
Direction of Synthesis:
R: replication in both directions with okazaki fragments, all DNA is replicated
T: single direction transcription in tightly regulated areas of genes
Proofreading:
R: DNA is proofread in replication and DNA repair happens constantly
T: RNA is not proofread, pol II makes several stats until a matching RNA is generated
When it Occurs:
R: All DNA is replicated in preparation for cell division
T: Gene specific promoters and transcription factors, cell and time specific
Enzymes Involved:
R: DNA polymerase
T: RNA polymerase II
Describe post-transcriptional RNA processing.
5’ Methylguanosine Cap:
- The first modification is the methylated 5’ cap on the mRNA
- A guanosine triphosphate (GTP) is added to the 5’ end of the mRNA via unusual 5’ to 5’ triphosphate linkage
- This GTP has a methyl group added to the 7 position of the guanosine base immediately after capping
- This occurs shortly after mRNA synthesis to protect the mRNA molecule from premature degedation by enzymes in the cell that degrade nucleotides + nucleases
3’ Polyadenylation:
- After the 5’ methylguanosine cap is added, two modifications occur in the same time period
- The first is 3’ polyadenylation:
- -> A different type of polymerase adds around 200 adenosines to the 3’ end of the mRNA immediately after it is cut from the RNA polymerase II; creating a structure called a poly(A) tail
- -> The poly(A) tail is essential for binding proteins that are necessary to transport the mRNA out of the nucleus and to start translation
RNA Splicing:
- The modification that occurs after the addition of the 5’ cap and 3’ polyadenylation is RNA splicing
- Genes are organized into exons and introns; introns are sequences of DNA that are not part of the mature protein, and fall between the exons
- Splicing is the mechanism that cells use to remove the introns from the mRNA sequence, leaving only the mRNA which codes for the protein
- Some genes will use alternate splicing to create multiple mRNA molecules from a single gene; this can cause exons to be skipped
- Most human genes can produce at least two different mRNAs through splicing - scientists still do not know if or when many of these alternate proteins are made
- Mutations at the splicing sites can lead to incorrect splicing and this may lead to proper protein translation
Transport Through the Nuclear Pore Complex
- RNA transcription occurs in the nucleus, since that is where DNA is stored
- At this point, post-transcriptional modifications are not complete however, RNA needs to exit the nucleus to be translated into protein in the cytoplasm
- A complex of proteins binds to the mRNA to assist in its transport out of the nucleus through the nuclear pore complex
- Some of these will be recycled and transported back into the nucleus, and others will help the mRNA in the process of translation
- The mRNA will not go back to the nucleus, but rather remain in the cytosol of the cell
How do 4 DNA bases give us the language for 20 different amino acids?
- It does so by using sequences of three RNA nucleotides, each of which corresponds to a specific amino acid or stop signal during protein synthesis
- One sequence of 3 nucleotides that codes for an amino acid is called a codon
Describe the importance of codons.
Each codon corresponds to one amino acid. There is also a codon that signals for translation to stop, which are called stop codons. It is important that codoms are three nucleotides long - this gives the cell enough possible combinations of nucleotides to account for every amino acid. In fact, it actually allows enough different combinations of the four nucleotides to have multiple codons coding for the same amino acid. One codon can only code for one amino acid. But many codons can code for the same amino acid.
What are the components of translation?
- Protein factors: initiation and elongation
- Initiation factors bind to the mRNA, including the methyl-guanosine cap on the 5’ end of the mRNA - these help the small ribosomal subunit identify the initiation site
- Elongation factors (EFs) are proteins that assist in elongation - some EFs form complexes to deliver the t-RNAs and the GTP energy source to the ribosome - Transfer RNA
- Each tRNA will recognize a codon and be attached to its accompanying amino acid
- -> The job of the tRNA is to deliver the correct amino acid to the growing peptide
- -> It does this by containing a complimentary sequence to the codon, termed the anticodon
- -> tRNAs are also bonded to an amino acid
- The bond holding the amino acid to the tRNA provides the energy to make the new peptide bond on the growing amino acid chain - Ribosome
- The ribosome is composed of a large and small subunit, both made of ribosomal RNA
- The small subunit is responsible for binding to the mRNA, while the large subunit has four important sites where the peptide is built
- -> A site: where tRNAs first attach to the ribosome
- -> P site: where newly arrived amino acid is removed from its tRNA and added to the growing peptide by a peptide bond
- -> E site: where the spent tRNA is ejected from the ribosome
- -> Finally, there is the polypeptide exit tunnel, where the peptide is guided and is ultimately released - Messenger RNA
- Messenger RNA is delivered with some protein factors from the nucleus to the cytosol
- Depending on the starting sequence of the messenger RNA, it will either stay in the cytosol, or a signal sequence will direct it to the surface of the endoplasmic reticulum
Outline the steps of translation.
- Initiation
- First the small ribosome subunit attaches to the mRNA molecule near the methyl-guanosine cap at the 5’ end where initiation factors are bound
- In then crawls forward until the correct AUG sequence is found
- The initator tRNA then binds to it, and the large ribosomal subunit excloses the mRNA, with the initator tRNA in the P position - Elongation
- Elongation is a multiple step cycle in translation, and moves forward as a continuous loop
- Each cycle adds one amino acid to the growing chain of amino acids, called a peptide
- This is an energy-expanding process
- The steps are coupled with the addition of energy ruck molecules like GTP, to drive the process forward
- Previously used tRNA is ejected from the E site - an energized tRNA enters A site
- Peptidyl transferase, an enzyme within the large ribosomal subunit, moves the growing peptide in P site onto the tRNA amino acid on the A site
- The ribosome is ready for the next charged tRNA to enter the A site - Termination
- The growing peptide chain ends when the stop codon is reached
- They are not connected to a tRNA molecule unlike the other codons
- Rather, they attract release factors, which also fit into the A site of the ribosomes; it substitutes water for the amino acid to attach to the peptide in the P site, and leads to the production of a carboxylic acid and releases the peptide
- Finally, a ribosome release factor occupies the A site - this leads to the release of the large and small ribosome subunits from the mRNA, and these can be recycled for more translation
What could happen when an error is produced during DNA replication?
Errors during replication can result in a mutation in the DNA sequence which could lead to a mutated protein if it is not properly repaired.
Describe point mutations.
Single nucleotide is changed with the following outcomes:
- Silent mutation: the mutation does not cause the amino acid to change
- Missense mutation: the mutation does cause the amino acid to change
- Nonsense mutation: the mutation replaces an amino acid codon with a stop codon, ending translation, and preventing the production of the rest of the amino acid - this is very etrimental, especially near the start of a sequence
Describe insertion mutations.
An exra base pair is added to DNA. This shifts the 3-base pair reading frame down by one, which can alter every amino acid produced. A similar reading frame-shift effect is seen with two base pairs, but three will add a new amino acid.
Describe deletion mutations.
A base pair is removed from the DNA sequence. Like insertion, this alters the reading frame if not in multiple of three.
Describe the large scale deletion, insertion and recombination mutations.
This can involve entire chromosomes or just parts of chromosomes. These changes are often lethal.
Describe hydrophobic amino acids.
Hydrophobic amino acids are also called non polar. They can be, aliphatic, which indicates the R chain is a straight carbon chain, or aromatic, which indicates a circular carbon R chain with double bonds. The aliphatic hydrophobic groups are glycine, alanine, caline, leucine, isoleucine, methionine, and proline.
These amino acids are normally found in the core of the protein, or interacting with other hydrophobic molecules like fats or lipids in a membrane.
The structure of each amino acids will give the protein it is found in certain characteristics, for example:
- Glycine does not have an R group, but rather only a hydrogen - it is the smallest amino acid and adds flexibility to a peptide chain
- Alanine has a single carbon methyl group and is still small
- Proline, on the other hand, is rigid, since the end of its side chain forms a covalent bond with the nitrogen of the amino group
Describe aromatic amino acids.
The aromatic amino acids have ring structures with double bonds that have distinct properties associated with this chemical structure. These are very large, and the gain or loss of these amino acids can cause deformities in the protein structure.
Describe polar amino acids.
The polar hydrophillic side chains can form hydrogen bonds that stabilize proteins. These are more common on the outside of a protein.
- The polar amino acids are: serine, threonine, tyrosine, asparagine, glutamine and cysteine
- Tyrosine is an aromatic, polar amino acid
- Cysteine has a sulfide that can form a type of covalent bond with another cysteine called disulfide bond - these are significant for forming three dimensional protein structure
Describe charged hydrophilic side chains.
These amino acids carry a positive or negative charge, and are therefore hydrophilic. They are found on the outside of roteins where they can interact with water.
Describe peptide bonds.
Amino acids are linked together through the peptide bond.
- The carboxylic acid group of one amino acid and he amino group of the second amino acid undergo a dehydration step (a peptide bond)
- Peptide bonds occur only between these two groups, and not the R groups of the amino acids
- The amino acids can rotate around these bonds
- In a long chain of amino acids held together by peptide bonds, there will be two distinct ends: one with the amino group free, and one with the carboxylic acid group free
- -> These are called the amino terminus and the carboxy terminus
