DNA Replication (extras), RNA replication, Proteins and Protein Synthesis

What are possible causes of DNA double strand breakage?

What are possible causes of DNA double strand breakage?

-UV Radiation (from the sun)

-Chemicals

The primary function of RNA is

to create proteins via translation

protein aggregation

when misfolded proteins create plaques and due to their rigid structures, they become obstructions and contribute to many neurodegenerative diseases

-have extremely low free energy

• too much folding

What does transcription produce?

RNA strands complementary to parts of a DNA strand

what is the basic unit of a eukaryotic chromosome structure?

nucleosome

Label this diagram

The highest correlation to an individual getting cancer is

age

-steadily over decades, the body’s cells are developing mutations

• more than likely a portion of DNA repair enzymes are mutated: They can no longer detect when mismatching occurs

what is the strongest force that draws together oppositely charged atoms?

ionic bonds

any molecule with many polar groups will have a pattern of which types of charges?

a pattern of partial positive charges and negative charges on its surface

how could a large molecule, like a protein, bind to another large molecule?

through non covalent bonds on the surface of each molecule

hydrophobic force

forces hydrophobic portions of dissolved molecules together to minimize their disruption of hydrogen bonded networks of water

-promote molecular interactions and the construction of cell membranes

• largely from lipid molecules with long hydrocarbon tails

how are organic molecules important in cells?

-some are used as monomer subunits to construct the cell’s macromolecules—proteins, nucleic acids, and large polysaccharides

-others serve as energy sources, being broken down and transformed into other small molecules

most covalent bonds that link together the subunits in a macromolecule allow _____ of the atom that they join, allowing the polymer to have great flexibility and allowing the macromolecule to adopt an unlimited number of _________

rotation, conformations

noncovalent bonds can stabilize associations between any two macromolecules, as long as ______________

their surfaces match closely

-these matches allow macromolecules to be used as building blocks for the formations of larger structures

-mediate interactions between macromolecules and other molecules

what types of bonds are needed to form a macromolecular assembly, like a ribosome?

both covalent and noncovalent bonds

how do enzymes promote intracellular chemical reactions?

by facilitating the molecular rearrangements that support cell function

-they bind to the reactant molecules and hold them in such a way as to make the chemical bond-breaking and bond-forming processes take place more quickly

what do protein transporters and channels in the plasma membrane do?

they control the passage of nutrients and other small molecules into and out of the cell

why does each polypeptide chain have directionality?

because the two ends of each amino acid are chemically different (one has an amino group, NH3+/NH2, and the other has a carboxyl group, COO-/COOH), each polypeptide chain has directionality

what are the three hydroxyl amino acids and their side chains?

serine (ser)- uncharged polar

threonine (thr)- uncharged polar

tyrosine (tyr)- uncharged polar

amino acids with nonpolar side chains

alanine

glycine

valine

leucine

isoleucine

proline

phenylalanine

methionine

tryptophan

cysteine

amino acids with negatively charged side chains

aspartic acid

glutamic acid

amino acids with positively charged side chains

arginine

lysine

histidine

what are the conformations of proteins controlled by?

sets of weak noncovalent bonds that form within the proteins

-the noncovalent bonds that help proteins fold up and maintain their shape are intermolecular forces

• it takes many noncovalent bonds to hold two regions of a polypeptide chain tightly together

proteins fold into a conformation of _____ energy

lowest

-each protein has a particular 3d structure, determined by the order of the amino acids in its polypeptide chain

a protein generally folds into the shape in which its free energy is _______

minimized

β sheets form the basis of many proteins, including amyloid structures that cause

disease

-neurodegenerative diseases like alzheimers, parkinsons, huntingtons

what brain eating disease can misfolded proteins cause?

prions
-mad cow disease

-Creutzfeldt Jakob disease

nuclease (DNAase)

breaks down nucleic acids by hydrolyzing bonds between nucleotides

protease (BACE/PS1)

breaks down proteins by hydrolyzing peptide bonds between amino acids

polymerase (DNApol)

catalyzes polymerization reactions like the synthesis of DNA and RNA

aggregates have a lower free energy, so theyre

more stable

synucleinopathies

-diverse group of neurodegenerative disorders that share a common pathologic lesion composed of aggregates

-parkinsons

-dementia

-multiple system atrophy

disulfide bonds

the most common cross-links in proteins (S-S)

-are formed before a protein is secreted by an enzyme in the endoplasmic reticulum

-are covalent bonds

-do not change a protein’s conformation, but instead act as an “atomic staple” to reinforce the conformation, stabilize proteins, and link protein chains

ligand

any substance that is bound by a protein

-a protein is able to bind to a ligand due to the formation of a set of weak, noncovalent bonds and favorable hydrophobic forces

• the ligand must, therefore, fit precisely into the protein’s binding site so that a large number of noncovalent interactions can be formed between the protein and the ligand

how are RNA strands produced?

through transcription

-transcription produces RNA strands complementary to parts of a DNA strand

• the template DNA strand runs 3’ to 5’ because RNA runs 5’ to 3’

what are the essential three types of RNA?

messenger RNA (mRNA)- codes for proteins

ribosomal RNA (rRNA)- forms the core of the ribosome’s structure and catalyzes protein synthesis

transfer RNA (tRNA)- serves as adaptor between mRNA and amino acids during protein synthesis

what are some differences between RNA and DNA molecules?

RNA

-single stranded

-ribose sugar

-uracil nucleotide

-hydroxyl group

DNA

-double stranded

-deoxyribose sugar

-thymine nucleotide

-methyl group

messenger RNA (mRNA)

type of RNA which codes for proteins

ribosomal RNA (rRNA)

form the core of the ribosome’s structure and catalyzes protein synthesis

microRNA (MIRNA)

regulate gene expression

transfer RNA (tRNA)

serve as adaptors between mRNA and amino acids during protein synthesis

-anticodons land on complementary nucleotides (codons) on the mRNA and release amino acids, which, through covalent bonds, bond together to form polypeptide chains

-each tRNA is specific for the amino acid it is carrying

How do different RNA molecules make interactions?

via H-bonds as seen during protein translation

how is the single stranded nature of RNA an advantage?

the single-strandedness makes it easier to fold in different conformations

-it can even make conformations within itself

how does RNA folding work?

bases find complementary sequences within the same molecule to form certain conformations

RNA polymerase I

most rRNA genes

RNA polymerase II

all protein coding genes, miRNA genes, plus genes for other noncoding RNAs

RNA polymerase III

tRNA genes

5S rRNA gene

genes for many other small RNAs

label

What are the big differences between DNA and RNA synthesis?

-unlike DNA polymerase, RNA polymerase does not need a primer

-unlike DNA polymerase, RNA polymerase does not need a helicase to open the double DNA

-unlike DNA polymerase, RNA polymerase does not copy the entire DNA, but only selected regions, called genes

what about RNA polymerase is needed for an effective transcription?

-RNA polymerase needs to recognize the gene to be described

• It needs to recognize which strand the gene of interest is found

• It needs to find the starting point of the gene of interest

-RNA polymerase needs to bind tightly to DNA

True or False: The amount of RNA polymerase in a cell’s nucleus is very high, and it could start transcription every time one molecule, or RNA polymerase, "collides” with DNA

true

-the abundance of RNA polymerase in the nucleus of a cell is critical for ensuring the timely and efficient transcription of genes, allowing cells to respond rapidly to changing conditions and carry out essential cellular processes.

promoter

helps with the transcription of RNA by “promoting” genes to be generated

• a region of DNA upstream of a gene where relevant proteins (such as RNA polymerase and transcription factors) bind to initiate transcription of that gene

• located at the 5’ end of the region

transcription factors

proteins involved in the process of converting, or transcribing, DNA into RNA

• Transcription factors include a wide number of proteins, excluding RNA polymerase, that bind the to promoter and help RNA polymerase bind to DNA

Where does RNA polymerase attach to in DNA?

in a region close to the promoter

-RNA polymerase synthesizes an RNA strand complementary to a template DNA strand

• It synthesizes the RNA strand in the 5' to 3' direction, while reading the template DNA strand in the 3' to 5' direction

-promoter is never copied by RNA

How does transcription start/stop?

through specific DNA sequences which allow the beginning and end of transcription

How does transcription begin?

in eukaryotic cells, transcription starts with “bending” DNA structure

-It has a promoter sequence with the nitrogenous bases of TATAAAA, also known as the TATA box, which starts the transcription process when the transcription factor of TFIID binds to it to start the initial assembly on the promoter

• the TATA box is located 25 nucleotides upstream of the site where transcription begins

How does TFIIB (transcription factor) play a role in RNA transcription?

TFIIB binds to TFIID, optimizing the possible interaction of RNA polymerase with transcription factors and DNA because TFIIB and TFIID are transcription factors

what are the roles of transcription factors in protein synthesis?

TFIIE (dont need to memorize) facilitates the assembly of TFIIH (also dont need to memorize), which unwinds DNA and modifies RNA polymerase for activity

-TFIIF prevents random binding of RNA polymerase to nonspecific DNA regions

How does RNA polymerase utilize ATP in transcription?

Using energy from the hydrolysis of ATP, the polymerase “closes” around the unwinded DNA to initiate transcription

-process releases pyrophosphate on the C-terminal domain

what are the three phases of RNA synthesis?

initiation

elongation

termination

initiation process of RNA synthesis: transcription

RNA polymerase binds to the promoter region of DNA

-phosphorylation of RNA polymerase allows RNA processing proteins to assemble on its tail

• phosphorylation step dissociates the RNA polymerase II from other proteins present at the start point of transcription

• it also allows a new set of proteins to associate with the RNA polymerase tail that functions in transcription elongation and pre-mRNA processing

elongation process of RNA synthesis: transcription

-when the DNA strands separate, RNA polymerase adds nucleotides to the template (antisense) strand of the DNA to make the mRNA

• RNA polymerase reads the DNA strand 3’ to 5’

termination process of RNA synthesis: transcription

-the RNA polymerase molecule and mRNA strand separate from the DNA template strand

-the poly-A polymerase enzyme caps the 3’ end of the mRNA strand, known as the poly-A tail (AAAA), through polyadenylation

-at the beginning of transcription, the 5’ end was also capped by a methyl-guanosine cap, which stabilizes the pre-mRNA and protects it from being degraded by other enzymes

DNA is used to create a _____ strand

pre-mRNA

introns

longer sequences of nucleotides which do not code for anything

-they are removed through RNA splicing

• the phosphodiester bond between the first exon and the first intron is broken

• a characteristic only present in eukaryotic cells

exons

shorter sequences of nucleotides used to synthesize proteins

translation

the process of taking the information stored on an mRNA strand to construct a protein

where does the mRNA strand go after it synthesizes in the nucleus?

it leaves the nucleus and enters the cytosol where it interacts with a ribosome

-it interacts with a tRNA molecule within the ribosome

initiation process of translation

-the charged tRNA molecule with the UAC anticodon and methionine amino acid enters the ribosome at the P site, where the peptide bonds are formed

• initiation factor gives the small subunit the chance to find a start site before it releases the large subunit

-In the P site, there is a tRNA with the proper AUG codon to attach to it

• now, the large subunit joins the complex

elongation process of translation

-another tRNA molecule enters the ribosome through the A site and the large subunits catalyzes two reactions:

• a bond is broken between the tRNA and the amino acid

• a covalent bond forms between the two amino acids: the one on the P site and the one on the A site

  • The large subunit has peptidyl transferase activity only

-as the process continues, the tRNA on the P site will move to the E site, losing its amino acid, and ejecting from the ribosome

• a growing chain of amino acids (polypeptide chain) forms, creating a protein

• ejected tRNA can be reused and reprocessed

termination process of translation

-when a stop codon enters the ribosome, it cannot be read by the tRNA synthetase but is instead read at the A site, causing a release factor to enter the A site, causing the small and large ribosomal subunits to disassemble

• stopping the growth of the polypeptide chain and completing the formation of a protein

-The protein leaves the ribosome and goes to the Golgi bodies, where it undergoes folding and conforms to perform a specific function

Lariat structure

the intermediate formed during the removal of introns from pre-mRNA

It is created when the spliceosome, a complex of proteins and RNA molecules, removes an intron from the pre-mRNA molecule

• it cuts the space after the first exon (5’ splice site) and before the next exon (3’ splice site)

  • spliceosomes recognize the splice sites on the pre-mRNA and snip them while joining the exons together

  • the intron debranches and then degrades

• the splicing creates a lariat, a lasso‐shaped structure formed from the fragment of RNA to be excised during the process of splicing out the introns

typically, different proteins are generated from different genes, but ________ splicing is when different proteins are generated from the same gene

alternative

-the genes can change which order of exons they want to include in the proteins (creating alternative mRNAs)

• typically, the order has to go exon 1, exon 2, but when proteins are generated from the same gene, the exons do not have to join consecutively, just sequentially

• they can join 1,2,3 or 2,3 or 1,2 or 2,3 but not 3,2,1 or 2,1

APP mRNA exists as three alternative spliced isoforms. What are they and how were they formed?

APP is a special protein that can have up to 18 exons

-APP 770 (the number referring to the number of nucleotides retained after splicing) and APP 751 are located in peripheral organs

-APP 714 (the predominant form) and APP 695 exist in the central nervous system

How do splicing factors affect proteins?

the type of splicing factors present determine the combination of a protein

what are the final protein products of APP?

APP 770

APP 751

and APP 695

true or false: ribosomes can all be reading the same mRNA and making different replications of it

true

large and small subunits

parts of a ribosome required for translation

-large is the upper part and small is the lower portion

-large is made of about 49 proteins and 3 RNA molecules

• the large subunit houses the tRNA binding site

-small is made of about 33 proteins and 1 RNA molecule

• the small subunit houses the mRNA binding site

codon

sequences of 3 nucleotides in the mRNA molecule which encode for amino acids

genetic information becomes protein through

codons and amino acids

tRNA synthetase

an enzyme that catalyzes the reaction to attach the appropriate amino acid onto its corresponding tRNA

-the anticodon is recognized by the synthetase and joins it with the proper tRNA

-once tRNA has amino acid attached, it is “charged”

-each tRNA synthetase interacts with both the amino acid and the tRNA carrying the matching codon

-when tRNA is ejected out through the E site, tRNA synthetase reprocesses it for re-use

which position of the triplet codon is the variable one?

the third one

GUA

GUC

GUG

GUU

this is called wobbling

How does the release factor work in rna synthesis?

-stop codons are not recognized by tRNA, so tRNA itself cannot stop translation however, the release factor can read the stop codon and act accordingly
-the release factor changes the activity of the peptidyl transferase by adding a molecule of water which releases the amino acid chain from the tRNA

-after the amino acid chain has been released, the ribosomes and the mRNA dissociate to begin a new reaction

reading frame

a way of dividing the sequence of nucleotides in a nucleic acid

• made of a set of consecutive, nonoverlapping triplets

• when reading frames shift, it changes the identity of the codon

frameshift mutation

the insertion or deletion of nucleotide bases in numbers that are not multiples of three

-affects the sequencing of the entire mRNA

-can be dangerous

peptidyl transferase

an enzyme that catalyzes the addition of an amino acid residue in order to grow the polypeptide chain in protein synthesis

-located in the large ribosomal subunit

-removes the growing amino acid chain from the tRNA molecule in the A site of the ribosome and adds it to the amino acid attached to the tRNA in the P site

-afterwards, the large subunit translocates, moving the ribosome along the mRNA strand to continue the process of protein synthesis

The ribosomes cycle

association and disassociation of the subunits at the start and at the end of the translation process

• small ribosomal subunit

• initiator tRNA

• large ribosomal subunit

• aminoacyl-tRNAs

• polypeptide

• mRNA start/stop codon

polyribosome/polysome

about 20 ribosomes read a single mRNA

-the small subunit binds to the 5’ end of the mRNA and then moves downstream along the mRNA until it reaches the AUG start codon, and then the large ribosomal subunit attaches to complete the ribosome

-allow the cell to make much more protein from a single mRNA

How does the COVID-19 virus manifest in the body?

-it attaches its spikes (S) to a host cell

• the cell ingests the virus through endocytosis

• the spike protein has a high mutation rate

what do antibodies recognize?

proteins

-specifically with COVID, antibodies recognize the spike protein, enabling them to bind to the surface of the virus, preventing the virus particles from attaching to the host cell

-if the protein makes its way into the host cell, antibodies can prevent the virus from releasing its genome into the host cell for replication

what are the four different types of immunizations?

-genetic code vaccines (mRNA)

• uses the genetic code of a virus to instruct the body to make antibodies for protection against the virus

-viral vector vaccines

• use a harmless virus as a delivery vector to introduce antigens into the body, which stimulates an immune response

-subunit vaccines

• a type of vaccine that contains only specific antigens or parts of pathogens rather than the whole pathogen itself

• The antigens stimulate an immune response against the pathogen without causing the disease

-weakened/inactive vaccines

• live attenuated forms and killed forms of the virus are used to generate antibody production in cells

how does the COVID vaccine work?

-the mRNA in the vaccine teaches the cells how to make copies of the spike protein

-when the covid spike protein makes its way towards the cells, the immune system will react to the presence of the cell

-antibodies are generated by the immune system to target the covid spike protein

nucleosome

made up of histones

-provides structural support for a chromosome

• histone octamer (8 histone core)

-wrapped in DNA

-subunit of chromatin

• “beads on a string”

TFIID

transcription factor involved in the recognition of the core promoter sequences

TFIIB binds to TFIID, optimizing possible interaction of RNA polymerase with TFs and DNA

TFIIB

interacts directly with the TATA-binding protein (TBP) and helps to recruit RNA polymerase II into the initiation complex

TBP

tata binding protein

how do the non-main TFs help in gene replication?

they change the shape of DNA to help it be read better by RNA polymerase