module 1 Flashcards
What are the 4 classes of biomolecules
lipids, proteins, carbohydrates, nucleic acids
central dogma of biology
DNA (DNA replication) (transcription) -> RNA (translation) -> Protein
role of lipids
energy storage, cell structure (membrane), signal molecule
roles of proteins
enzymes, build tissues/muscles, hormone production
roles of carbohydrates
fuel source, cell identification
roles of nucleic acids
provide genetic information for processes such as DNA replication; transcription; translation.
differentiate between nucleotide and nucleoside
nucleotide:
- made up of a sugar (ribose or deoxyribose), nitrogenous base, and 1+ phosphate group
nucleoside:
- made up of a sugar (ribose or deoxyribose) and a nitrogenous base (no phosphate group)
differentiate between RNA and DNA
RNA
- contains a ribose sugar
- contains information to form a protein
- single stranded
- contains the pyrimidine Uracil instead of Thymine
DNA
- contains a deoxyribose sugar
- contains the information to form RNA therefore forming a protein
- double stranded (double helix)
- contains the pyrimidine Thymine instead of Uracil
Desribe the structure of nucleic acids
Made up of monomers called nucleotides:
- contain a pentose sugar (deoxyribose in DNA ribose in RNA)
- contain a nitrogenous base (Adenine, thymine/uracil, guanine, or cytosine)
- contain a phosphate group in 5’ carbon
How does the double helical structure of DNA play a role in semiconservative replication?
role in accurate transmission and preservation of genetic information?
When DNA replication occurs, each parent strand is separated and in the end, 2 daughter strands are created joining with each parent strand. This is semiconservative because each new DNA double helix contains a parent strand and newly made daughter strand. Genetic information is perserved because when DNA is replicated, the newly made DNA will always contain a parent strand.
What are the enzymes and proteins involved in DNA replication? Describe their roles.
- DNAa protein: melts/opens the DNA at the origin of replication using ATP for energy
- helicase: unwinds the DNA double helix
- SSB proteins: binds to single strands to prevent the DNA from reattaching
- topoisomerase: binds to strand and prevents supercoiling
- primase: applies primers (a stretch of complementary RNA bases 5-10 nucleotides long) to indicate where replication should start
- DNA polymerase III: applies complementary bases (nucleotides) to the DNA strand (starts at 3’ end of RNA primer)
- DNA polymerase I: replaces the RNA primers with DNA, adding nucleotides to 3’ end of fragments (in lagging strand)
- DNA ligase: seals the okazaki fragments in the lagging strand
Explain the steps involved in DNA replication
- a helicase protein unwinds the DNA double helix
- SSB proteins bind to each strand to prevent them from re-attaching
- Topoisomerase also binds to each strand to releive the tension and prevent supercoiling
- Primase synthesizes RNA primers using the DNA parental strand as a template, this primer is usually 5-10 nucleotides long
- DNA polymerase III does complementary base pairing (with parental strand) adding DNA nucleotides, starting at the 3’ end of the primer.
- LEADING STRAND COMPLETE
- Primase adds fragments of primers to the parental strand
- DNA polymerase III applies complementary base pairing in between each fragment of primers.
- DNA polymerase I replaces the RNA in the primers with DNA
- DNA ligase seals the gaps between the nucleotides from DNA poly III and DNA poly I.
Describe the process of prokaryotic transcription
differentiate between eukaryotic transcription and prokaryotic transcription
prokaryotic
- occurs in cytoplasm
- has no post-transcriptional modifications
eukaryotic
- occurs in the nucleus
- post-transcriptional modifications occur
- requires transcriptional factors
- requires RNA polymerases I, II, and III
Differentiate between the 3 types of RNA
their roles & differences in their post-transcriptional modifications
tRNA
- carries amino acids during translation, using complementary base pairing
- Addition of CCA nucleotides at 3’ end
- cleavage and the bases are modified
rRNA
- makes up the ribosomes (along with proteins)
- pre-rRNA gets modified and cleaved: methyl groups added in modification and cleavage occurs (think intron splicing)
mRNA
- contains information for protein synthesis
- a 5’ methylguanylate cap gets added
- a 3’ poly-A-tail gets added (250 adenylate nucleotides)
- introns are spliced
What are the components required in protein synthesis/ translation?
- amino acids
- tRNA
- mRNA template
- Ribosome
- release factor
- initiation/elongation protein factors
- amino acyl tRNA synthases
- Energy (GTP and ATP)
What are the roles of the different RNAs in protein translation?
rRNA
- builds the ribosomes
tRNA
- brings the amino acids to the ribosome
mRNA
- contains the information for what amino acid to bring (code) therefore dictates what protein is being built
What is the role of protein synthesis/ translation in storage and expression of genetic information?
genetic information is stored and expressed through an mRNA strand. The sequence of an mRNA strand is used to create the protein during translation.
describe the relationship between the genetic code and amino acids.
During translation, the genetic code is expressed through an mRNA moleucle. The genetic code, which is the information contained in the DNA, is expressed as an amino acid sequence during translation.
What are the mutations that can occur during translation?
Point mutation: a single nucleotide is changed
- missense mutation: nucleotide change causes a different amino acid to be coded
- nonsense mutation: nucleotide change causes a termination codon
- silent mutation: nucleotide change codes for the same amino acid
Trinucleotide repeat expansion: repeated copies of codons results in amplification of codons (too many copies of 1 amino acid)
Splice site mutation: mutation at splice site junction, changes how introns are removed from pre-mRNAs
- leads to proteins that are too long/short
Frameshift mutation: insertion/deletion of 1-2 nucleotides shifts the reading frame
What are the steps involved in translation?
Initiation
1. ribosome scans the mRNA template until it reaches the right AUG (start codon)
2. Initiation factors bind to small ribosomal subunit , forming 30s initiation complex, large subunit joins
Elongation
1. AUG moves to P site, tRNA brings next amino acid to A site, peptide bond forms between amino acids
2. peptidyl transferase catalyzes the bond formation
3. Translocation (A –> P –> E) requires energy, GTP
Termination
1. Stop codon is reached (UAA, UAG, UGA)
2. release factor binds (not an amino acid) and the peptide is released
3. entire complex dissociates
What occurs during post-translational modification? What is the role of post-translational modifications?
Post-translation modifications include covalent attachments (methylation, hydroxylation, phosphorylation, acetylation) or post-translational cleavage.
Role: mediates folding, stability, and localization
What are restriction endonucleases?
Enzymes produced by bacteria, cleave double stranded DNA at (specific) palindromic sequences, used for cloning.
What is cloning? explain the process.
introducing foreign DNA into a host cell (bacteria) using vectors, to amplify the DNA sequence. Used for PCR, to make DNA libraries, and amplify DNA.
- DNA is cleaved with specific restriction endonuclease
- each fragment is joined to a cloning vector (hybrid DNA molecule)
- “hybrid DNA molecule” introduced to a bacteria cell, replication of clones occurs
- Cloned DNA releases from cloning vectors using REs are isolated and analyzed
Differentiate between cDNA and gDNA libraries. When would each be used?
cDNA stores the mRNA sequence (expressed genes) whereas gDNA stores the whole gene sequence. gDNA can be used to identify the presence of a gene and cDNA can be used to identify genes expressed in cancer or other diseases.
- in cDNA libraries, the mRNA used as a template undergoes reverse transcriptase
Explain the process of PCR. When would it be used?
- Denaturation: the DNA is heated at 95˚C for 1-2 mins to separate DNA strands
- Annealing: reaction cooled to 70˚C for 1 min to allow primers to anneal to flanking regions of DNA (where promoter/primer is)
- Extension: DNA polymerase and dNTPs initiate synthesis of 2 new chains complementary to original DNA strands
Used for:
- comparison of normal gene with mutant
- Diagnose viral infection (COVID-19)
- forensic analysis of DNA samples
- prenatal diagnosis and carrier detection of cystic fibrosis (CF)
Explain the process of microarrays. When would this be used?
Microarrays view gene expression under different conditions
- mRNA is converted to cDNA (reverse transcriptase) and labeled with a fluorescent tag
- mixture exposed to gene chip that contains many tiny DNA spots (each correspond to a specific gene)
- cDNA binds to matching DNA spots making fluorescence detectable
- amount of fluorescence proportional to amount of mRNA in sample
Application: used to determine patterns of gene expression in normal vs. cancer cells
Explain the process of western BLOTs. When would this be used?
- protein is extracted from the cell
- Protein is seperated by molecular weight and size using an SDS-page (gel electrophoresis)
- PVDF membrane placed on SDS gel to transfer protein on gel to the membrane
- An antibody that detects protein of interest is mixed into membrane to allow binding of protein. A band is only produced in region where antibody binds to protein.
- Detection signals used (colorimetric or chemiluminescent)
Application: compare expression
compare ELISA and western blotting. What are the applications of ELISA?
Same as western blotting but used to identify and quantify the presense of a protein of interest. Also it’s in a well not and SDS page.
Applications: detection and quantification of HIV antibodies
What would the sequence of the primary transcript be for the following DNA templatestrand?
5’ – ACGCTACTTGGACACTATA – 3’
template: 3’ ATATCACAGGTTCATCGCA 5’
coding:5’ TATAGTGTCCAAGTAGCGT 3’
mRNA:5’ UAUAGUGUCCAAGUAGCGU 3
are these restriction endonuclease sites?
5’- GGCGCC-3’
5’- ATGGTA- 3’
5’- ACTAGT- 3’
5’- CTCT- 3’
yes
no
yes
no
What is southern blotting used for?
to detect whether a specific DNA sequence is present in a DNA sample
