DNA to RNA to Protein

Nucleotide structure

Responsible for transmission of genetic information, preservation of genetic information, roles of nucleosides and nucleotides, energy storage, energy transmission, signaling, act as antioxidants

ribose

deoxyribose

purines

A and G

pyrimidines

C, T, and U

nitrogenous bases

Attach to the carbon 1'

Phosphate groups

One, two, or three attached to the carbon 5'

Nucleoside

Sugar and a base, but no phosphate group

Ribonucleic acids

- Nucleotides with ribose sugars
- Many can be attached to each other to make RNA

Deoxyribonucleic acids

- Made from nucleotides with deoxyribose
- Form DNA

Sugar-phosphate Backbone

The covalent bonds that connect nucleotides join the phosphate of one nucleotide to the sugar of another

The double helix

- Formed by phospho-diester bonds and form by the loss of two phosphates with each attachment
- Nitrogenous bases are hydrophobic aromatic rings that want to be away from water
- Charged phosphates are hydrophilic and very happy to be near water

Double stranded property of DNA

- Formed by intermolecular interactions = hydrogen bonds between nitrogenous bases
- Base pairing: purine always pairs with the pyrimidine

Antiparallel

- Two strands run in opposite directions
- As a result, the enzymes will operate in opposite directions

DNA denaturation

The process of separating two DNA strands into single strands

Helicase

- Separates DNA strands
- Breaks hydrogen bonds between nitrogenous bases

DNA denaturation by temperature

- Will be complete at 95 ˚C
- G-C hydrogen bonds break at higher
- A-T break hydrogen bonds at lower

Reannealing

- The process of two single DNA strands that have been separated by helicase or heat coming back together to form the original double stranded DNA
- Favored by hydrogen bonding and hydrophobic regions that want to stay away from water

Hybridization

- When a complementary (sometimes shorter or tagged) strand of DNA is annealed (paired) to another of interest
- If the sequences are not complementary, this will not occur

Central Dogma

Explains the flow of information in all biological systems

Anticodons

- Found on tRNA
- During the process of translation, each codon is recognized by a complementary one of these

Codons

- 64 possible
- Each is specific and unambiguous, corresponding to precisely one amino acid
- But the genetic code is degenerated: has built in redundancy so that more than one of these can correspond to the same amino acid
- How we have only 20 amino acids

Wobble

- Due to degeneracy of genetic code
- When multiple codons code for the same amino acid, most often those codons those codons share the same first two bases
- The third nucleotide of the codon is the variable one
- Due to the tRN anticodon literally wobbling in matching the final nucleotide to the mRNA codon
- An evolutionary development that protects us against mutations
- If a mutation occurs in the wobble position, or a slightly wrong tRNA is brought in, that change is likely to be a silent mutation (no effect on the polypeptide sequence)

Point mutation

A mutation affecting only on nucleotide in a gene sequence

Missense mutation

- A mutation where one amino acid is substituted for another
- Alter the primary amino acid sequence of the protein
- We call them expressed mutations

Nonsense mutations

- Are even more serious than missense mutations
- They change a codon from an amino acid to code for a premature stop codon instead
- Also known as truncation mutations

Silent Mutation

does not affect the protein

Frameshift Mutation

involves the deletion or insertion of a base which results in a change to the gene’s reading frame

Deleterious mutation

genetic variation that is known to be associated with an increases risk of disease

AUG

- Start codon on the mRNA
- Codes for methionine
- It signals where to start translation of mRNA to protein

Stop codons

UAA
UGA
UAG

Initiation (transcription)

- The start of transcription
- involves the:
promoter region of the gene
Transcription factors
TATA box (euk)
The Pribnow box (pro)
Helicase
RNA pol

Promoter region of the gene

RNA pol binds onto the DNA many base paris in advance of the actual start site of transcription

Transcription factors

proteins that may bind to the promoter region to help signal that RNA pol should bind there as well

TATA box

- Most euk and pro genes have a sequence of bases that is rich in T and A nucleotides that RNA pol recognizes and binds onto
- Is located 25 to 35 base pairs upstream of the actual start site

The Pribnow box

- In prokaryotes
- About -10 base pairs from the start site
- Not a TATA box
- rich in Ts and As and also the site of RNA pol binding

Helicase

- Unwinds the DNA, breaks the H-bonds between nucleotides
- A subunit of RNA pol itself

RNA pol

- Reads the base of the gene and attaches a complementary RNA nucleotide onto the DNA
- Keeps reading the DNA and matching the correct RNA base pair, while simultaneously catalyzing a new phosphodiester backbone bond between the growing RNA strand and the next RNA nucleotide

elongation (transcription)

involves: RNA pol, Sense strand/coding strand, Antisense strand/template strand

Sense strand/coding strand

The strand of DNA that looks exactly like the synthesized strand of RNA

Antisense strand/template strand

The strand of DNA that is the opposite of the synthesized strand of RNA that was read by RNA pol to create complementary RNA base pair match

Termination in prokaryotes (transcription)

Rho independent or Rho dependent

Rho

A protein that can bind on the synthesized strand of RNA

Rho-dependent

- Rho binds to the synthesized strand of RNA, which introduces steric strain which tugs n the RNA and pulls it away from the RNA pol
- The RNA is now ready to be immediately translated and the RNA pol finishes the job by sealing together the DNA strands and detaching to go find another gene to transcribe

Rho-independent

- G-C rich sequence in DNA is then synthesized as a G-C rich sequence of mRNA
- They create a hairpin loop (fold) that causes steric strain, tugging RNA away from the DNA

termination in eukaryotes (transcription)

includes 5' modification, 3' poly A tail, and splicing

5' modification

- The addition of a methylguanosine cap (guanine nucleotide attached backwards)
- Prevents the degradation of the mRNA transcript
- Prevents it from fitting in the active site of the exonuclease that would have degraded the mRNA transcript

3' poly A tail

- Long sequence of adenine residues is added to end of the mRNA
- Prevents degradation
- The exonuclease would have to break away each adenine one by one, which would take more time before it reaches the signaling the part of the mRNA
- Longer = more iterations of translation of the mRNA, signal stays around longer
- Shorter = message lasts around less time

splicing

Excising introns: introns removed before the mRNA exits the nucleus

rRNA

- Structural RNA that composes the large and small subunits of the ribosomes themselves
- Do not code

Structure of ribosomes

- The small subunit binds first
- The large subunit contains the A, P, and E sites

Initiation (translation)

The small subunit binds to the mRNA before the start codon

initiation in Prokaryotes

The small subunit binds to the shine-dalgarno sequence

initiation in Eukaryotes

The small subunit binds to the 5' cap from the post-transcriptional modification

translation

- doesn't start until AUG is recognized by the small subunit
- Then the large subunit initiates the binding at the A, P, and E sites

Elongation (translation)

An amino acid bound to the tRNA binds to the A site and is moved over to the P site via peptidyl transferase

Termination (translation)

- Determined by stop codons
- Release factor comes and binds to the A site to release the ribosome from the mRNA and the tRNAs

Chaperone proteins

Condense the polypeptide into a 3D shape so that it becomes a recognizable protein

Chromatin structure

Histones

- Protein that DNA molecules are wound around
- Around 200 bp of DNA are wounds around one complex

Nucleosome

The complex of a histone protein wrapped by 200 bp of DNA

Euchromatin

- When the nucleosome complexes look like beads on a string
- Allows the DNA molecule itself to be easily accessed
- Crucial to allow RNA pol to bind to DNA and initiate transcription

Heterochromatin

Further compacted euchromatin

DNA methylation

- Way to alter the structure of chromatin
- Cytosine and adenine bases in DNA can be methylated
- Hinders the ability of RNA pol to transcribe and is also associated with more compacted DNA

Histone acetylation, methylation, and phosphorylation

- Decrease their affinity for DNA and makes the DNA molecule more accessible
- Conversely, removing these groups makes the chromatin more compact so it's less available for transcription

Histone displacement

- Slide apart
- Allow the DNA sequence between them to be accessed more easily

Transcription factors

Bind to response elements (specific DNA sequences) which are located in the promoter region of every gene

General transcription factors

- Serve crucial roles in transcription machinery
- Recruit RNA pol and unwind DNA helix

Selective transcription factors

- Bind to specific response elements that are found in only some genes
- The response elements they bind to are often found hundreds of bases away from the promoters of the gene (enhancers)
give the cell an excellent way to regulate which genes are expressed
- In the presence of specific signals, they will bind to their response elements in the enhancer region and either promote or inhibit the transcription of those genes

Post-transcriptional regulation

The nucleus can regulate which mRNA transcripts and how many are exported from the nucleus

Untranslated regions or UTRs

- On both the 5' and 3' end of the mRNA transcript
- Generally not translated into protein
- Serve critical functions in regulating translation
- 5' UTR: ribosome relies on it to bind to to begin translation
- 3' UTR: often forms secondary structures that help stabilize the transcript or are recognized by specific enzyme that degrade the transcript

Oncogenes

- Promote cell division
- Overexpressed in cancer cells
- More cell division → tumor
- Normally are suppressed in nondividing cells, preventing tumor formation
- Many kinds of cancers are the result of the overexpression of these genes

Tumor suppressor genes

When incorrectly suppressed, cells either divide more frequently (increase likelihood of tumor formation) or accumulate DNA damage (making more susceptible to tumor-promoting mutations)

Gatekeeper

- Directly supports growth
- Cell cycle regulator genes
- Checkpoint control genes
- Apoptosis related genes

Caretaker

- Maintain overall genetic stability
- DNA repair proteins

PCR

- Use a set of primers (complementary DNA sequences that we design) to locate sequences of DNA on the target site
- DNA strands go through cycles of denaturing, hybridization, and replication

Solution used for PCR

Contains DNA fragments, dNTP (ATCG), enzymes, and primers

Denaturing step of PCR

- Uses incredibly high temperatures to heat up solution and separate double stranded DNA sequences into two parental single-stranded sequences
- To about 94-96 ˚C to break down hydrogen bonds

Hybridization step of PCR

- Solution is cooled
- DNA strands will anneal to the primers to form a short DNA hybrid

Replication step

- DNA pol comes in to extend the hybrid DNA
- Reaction mixture will be heated to at least 70˚C in order for Taq pol to function properly
- Sequences then form dual stranded hybrid that can then be broken apart and the cycle can be repeated with a new template

Taq polymerase

- Used in PCR to make sure polymerase doesn't denature at very high temperatures
- From bacteria that live in volcano vents on the ocean floor

Gel electrophoresis

- Figure out how many base pairs long a segment of DNA is
- Works by moving fragments of DNA through an agarose gel plate
- Smaller pieces of DNA can move faster than larger ones
- DNA fragments all move forward because of the negatively charged phosphodiester backbone
- Can compare the fragments' movement to a DNA ladder (a separate solution of carrying fragments of DNA that we already know the exact sizes of
- Only allows us to determine the size of the fragment

Southern blotting

- Use probes to identify the target DNA after it has been run on a gel
- Useful for identifying if the target gene is in the sample
- DNA is blotted with a sheet containing a probe designed to hybridize with our desired DNA, after it's run through gel electrophoresis
- Nitrocellulose paper is used to block the DNA segment out of the gel, and onto the membrane
- A solution containing a fluorescently labeled hybridization probe for our target sequence is then placed on the membrane itself
- Since the primer only binds to a specific sequence that we design, the presence of a band after imaging the blot means that we've identified our gene of interest

Northern blotting

used to identify RNA

Western blotting

used to identify proteins

Subcloning

- The process of inserting genetic material into a plasmid, to make recombinant DNA, and is a form of cloning
- The problem is that there are many different plasmids that we can insert DNA sequences into
- Knock-in: pAcc-B plasmid

Restriction enzymes

Are site-specific, so enzymes will only latch onto a particular sequence of DNA on our plasmid