Biochemistry Flashcards
What is the difference between a nucleoside and a nucleotide?
Nucleoside- a 5 carbon sugar (pentose) bonded to a nitrogenous base
Nucleotide- A nucleoside + one or more phosphate groups attached to the 5th carbon of the pentose
What are the building blocks of DNA?
Nucleotides
What is the difference between the 2nd carbon on the pentose sugar on DNA and RNA?
RNA- has an -OH group
DNA- has an -H group because it is deoxyribose
On what carbon due RNA and DNA differ?
2 carbon of the pentose sugar ribose
How are Nucleic Acids and Nucleotides related?
Nucleic Acids (DNA and RNA) are long chains of nucleotides
In what direction is DNA always read?
5’ to 3’
Between what two carbon atoms of nucleotides does a phosphodiester bond form?
The 3’ of the top nucleotide and the 5’ of the bottom nucleotide
Why are DNA and RNA given an overall negative charge?
Because the phosphate groups are negative
If the nitrogenous base on a DNA molecule has two rings what are the possible nucleotides that make it up? What group of nitrogenous bases is this?
Adenine and Guanine
Both are in the Purine Group
If a nitrogenous base on an RNA molecule has one ring what are the possible nucleotides that make it up? What group of nitrogenous bases it this?
Uracil and Cysteine
Both are in the Pyrimidine Group
In DNA, what other nucleotide does Adenine bond with and how many hydrogen bonds does it make with it?
It bonds with Thymine and makes 2 Hydrogen Bonds
In DNA, what other nucleotide does Guanine bond with and how many hydrogen bonds does it make with it?
It bonds with Cysteine and makes 3 Hydrogen Bonds
Is the A w/ T or G w/ C base pairing stronger?
G with C because it uses one more H-bond
What must occur for DNA to be opened up for transcription and replication?
The Hydrogen bonds between the nucleotides must be broken
What does the process of reannealing DNA entail?
Two denatured strands of complementary DNA can be reannealed by slowly removing the denaturing condition such as heat. This brings the two strands back together
What are the proteins that DNA is folded around to form chromatin?
Histones
What are the various stages of DNA folding and consolidation?
- DNA double helix
- Nucleosomes (beads on a string)
- Supercoiling of nucleosomes
- heterochromatin
What is the difference between euchromatin and heterochromatin?
Euchromatin- unwound and loose chromatin
Heterochromatin- tightly bound chromatin
Do centromeres consist of euchromatin or heterochromatin?
Heterochromatin because they are tightly condensed
How does bacterial DNA replication differ from humans?
Bacteria have a large circle of DNA that is replicated starting at one origin of replicaton
What protein goes ahead of helicase and starts to unwind the DNA helix to prevent torsional strain?
DNA Topoisomerase
What protein is responsible for breaking the H-bonds between nucleotides to separate the 2 strands of DNA for replication?
Helicase
What proteins are responsible for synthesizing the new daughter strand of DNA by reading the parent strand?
DNA polymerases
What direction do DNA polymerases read? What direction do they synthesize daughter strands in?
They read from 3’ to 5’ direction
They then synthesize new DNA in the 5’ to 3’ direction
Why can only one of the parents strands of replicating DNA be effectively worked on by DNA polymerases?
Because the polymerase makes new DNA from 5’ to 3’ end and thus only one strand matches this pattern (the leading strand)
Why are Okazaki fragments created in the lagging strand?
Because the lagging strand cannot be read in a forward direction by the DNA polymerases and thus it works in a circular short segments
Which nucleic acid, RNA or DNA, can be synthesized without a primer? How does this effect replication?
RNA; this is why an RNA primer is added to DNA by primase to initiate replication
What proteins are responsible for the joining of Okazaki fragments on the lagging strand?
Ligases
What do oncogenes usually code for?
Cell-Cycle related proteins
What do antioncogenes usually code for?
Cell-Cycle inhibiting proteins (ex. p53 and retinoblastoma)
During S-phase, how does the cell regulate the errors associated with DNA replication?
The replicating DNA passes through part of the DNA polymerase to undergo proofreading
This is done by checking the stability of the H-bonds associated with the new DNA
During the G1 and G2 phase what are the two methods of repairing damaged DNA?
- Nucleotide Excision Repair- where proteins find errors in DNA and then endonuclease nicks the phosphodiester bond so that the nucleotide is removed and replaced correctly
- Base Excision Repair- the effected base is found and removed and then this indicates to an endonuclease that the nucleotide is removed and replaced correctly
What is a common way of creating many copies of a strand of DNA? Why is it useful?
Polymerase Chain Reaction (PCR)
It allows for DNA to be tested for genetic markers
Why do DNA primers used for PCR have high amount of Guanine and Cysteine nucleotides?
So that they are more stable
What two molecules can bind to histone proteins and what are there effects on the accessibility of DNA to transcription? What is the mnemonic to remember this?
Acetylation- occurs when acetyl groups bind to histone proteins and INCREASE accessibility
Methylation- occurs when methyl groups bind to histone proteins and DECREASE accessibility
Acetylation makes DNA Accessible
During transcription, what strand of the original DNA is identical to the new hnRNA except for the Uracil for Thymine exchange?
The Coding Strand
During Transcription, what strand of the original DNA is complementary and antiparallel to the new hnRNA?
The Template Strand
In what direction is new hnRNA transcribed?
From the 5’ to the 3’ end
What are the 4 types of RNA in the cells?
- mRNA- carries genetic information from DNA to ribosome
- tRNA- translates the mRNA information into an amino acid
- rRNA- function as enzymes made of RNA (ribozymes) and help the ribosome function
- snRNA- used in post-transcriptional modification of DNA by excising introns and exons
What is the difference between monocistronic and polycistronic mRNA? In what cell types do these separate RNA types exist?
Monocistronic- mRNA codes information for only one protein product; exists in Eukaryotes
Polycistronic- mRNA codes information for many proteins; exists in Prokaryotes
What does it mean for a tRNA to become activated?
It bonds with a specific amino acid
What is the name for the 3 letter codes of nucleotides that relate to a specific amino acid?
Codon
What are the 3 stop codons?
UAA
UAG
UGA
What is the start codon?
AUG
What is the first amino acid in every protein?
Methionine
What is the third (and often variable) base in a codon referred as?
Wobble Position
What two enzymes, used in replication and transcription, are important for the unwinding and breaking apart of the DNA double helix?
Topoisomerase- unwinding
Helicase- breaking apart
What are two names for the strand of DNA that is complementary and antiparallel to the new hnRNA strand?
Template or Antisense Strand
What is the protein that is responsible for transcribing DNA into hnRNA?
RNA Polymerase II
What are the specialized DNA regions to which RNA Polymerase II can bind to and initiate transcription?
Promoter Regions (ex. TATA box)
In what direction does RNA Polymerase II read the DNA antisense strand?
From 3’ to 5’ direction so that it can create hnRNA in the 5’ to 3’ direction
What is the difference between hnRNA and mRNA?
hnRNA is what is left immediately following transcription from DNA and has not undergone post-transcriptional modification
What are the 3 main events that occur during post-transcriptional processing?
- Splicing of Introns from Exons
- Addition of the 5’ GTP-cap
- Addition of the 3’ Poly-A tail
What is the way to remember that introns are removed from hnRNA and that exons are kept in mRNA?
Exons are Expressed (thus they have to be left in the mRNA)
What type of RNA is used in the processing of splicing introns?
snRNA that are a component of spliceosomes
Why is a GTP-cap given to the 5’ end of hnRNA in post-transcriptional modication?
This cap protects the 5’ end from degradation in the cytoplasm as well as serves as a target for ribosome binding
Why is a Poly-A tail given to the 3’ end of the hnRNA during post-transcriptional modication?
It serves as a protecting fuse for the hnRNA in the cytoplasm
Is the Poly-A tail of post-transcriptional modification added to the 5’ or 3’ end of hnRNA? What is the mnemonic for remembering this?
3 rats with poly-A tails
Where does the process of translation take place?
Ribosomes
What are the 3 steps of translation?
- Initiation
- Elongation
- Termination
What occurs in the process of initiation during translation?
The small ribosomal subunit binds to the 5’ untranslated region and the initiator tRNA binds to the start codon in mRNA
In what section of the Ribosome do new tRNA molecules enter and bind to the mRNA?
the A site
In what section of the Ribosome do the amino acids get added to a growing polypeptide chain?
the P site
In what section of the Ribosome do the deactivated tRNA molecules exit?
the E site
Proteins that are created to be secreted outside the cell contain signal sequences that direct the ribosome to move to what organelle?
The Rough ER
What processes allow a translated polypeptide chain to become a functioning protein?
Post-Translational modification
What are some examples of post-translational modification?
- Protein Super-folding (aided by chaperones)
- Cleavage events (ex. prohormones)
- Subunits coming together to form quaternary structure (ex. Hb)
What is the name for a group of genes, in prokaryotes, that are transcribed as a group?
Operon
What is the difference between an inducible and repressible system in operons?
Inducible- a protein binds to the regularly bound repressor on the operator site, this increases transcription (ex. lac operon)
Repressible- without a corepressor bound to it, a repressor cannot bind to the operator site, this increases transcription (ex. trp operon)
What is the difference between the lac and trp operons?
Lac- inducible operon
Trp- repressible operon
What are some of the methods that Eukaryotes control gene expression?
- Alternative Splicing
- Transcription Factors
- Enhancers
- Gene Duplication
- Histone Acetylation
- Histone Methylation
What is the role of transcription factors?
They are used in eukaryotes as a method of controlling gene expression by binding to specific regions of DNA and attracting proteins used in transcription
What is the role of Enhancers?
They are used in eukaryotes as a method of controlling gene expression by being activated by signaling molecules (cAMP, cortisol) and serve as a group of transcription factors
What is the role of gene duplication in controlling gene expression in eukaryotes?
Cells will create multiple copies of the same gene on the same chromosome to control how often it is transcribed
What would be the effect of histone deacetylation?
Decreasing gene expression
How does gel electrophoresis work?
A polyacrylamide gel is set up with a cathode (-) on one side and an anode (+) on the other
Proteins that are negatively charged will migrate toward the anode (+) thus separating them out by charge
What are the three main types of gel electrophoresis?
- Polyacrylamide Gel Electrophoresis (PAGE)
- SDS-PAGE
- Isoelectric Focusing
What is the limitation of a regular Polyacrylamide Gel Electrophoresis?
Proteins that are very massive but are heavily charged may migrate the same as a smaller but more charged molecule
Thus PAGE is best at analyzing proteins that are known to have similar sizes
How does SDS-PAGE differ from regular gel electrophoresis?
The Protein being analyzed is treated with SDS which disrupts all non-covalent interactions and binds to the protein with an equal mass to charge ratio
This allows SDS-PAGE to separate out proteins based on molecular mass alone
What is the process of Isoelectric Focusing?
A gel electrophoresis is set up with an acidic gel near the anode and a basic gel near the cathode
As the electric current is sent through the gel, proteins with a basic charge (+) will migrate toward the cathode and stop moving when they reach a gel pH that is equal to the proteins PI
When is chromatography favored over gel electrophoresis?
When large amounts of protein are being separated
Assuming the a group of proteins or DNA have similar net charges, what type of protein or DNA fragment would travel the farthest in a PAGE setup?
A small protein or DNA fragment
What are the different blotting techniques and what do they measure respectively?
Southern Blot- DNA
Northern Blot- RNA
O O
Western Blot- Protein
Mnemonic = SNOW DROP
Assuming a group of proteins have a similar size, what type of protein would travel the farthest in a PAGE setup?
The protein with the most negative net charge`
Is net charge a consideration for measuring results of a PAGE experiment done on DNA or RNA?
No; because DNA and RNA molecules of any length have proportional net charges
What is the difference between a regular SDS-PAGE and a reducing SDS-PAGE?
the reducing SDS-PAGE adds a chemical that breaks the cysteine-cysteine salt bridges left behind from the SDS treatment
What is a native-PAGE?
Running a PAGE setup with the natural protein (regular and untreated)
What is the general process of a Northern or Southern Blot?
RNA is separated using a PAGE setup and then added to nitrocellulose and UV light to immobilize it. Then a reporter (marker) RNA fragment is added to label a specific part of the RNA you are analyzing
How is a Western Blot different from a Norther and Southern Blot?
A Western Blot analyzes proteins and thus cannot use RNA probes to mark a specific segment
Instead a Western Blot uses a Primary (for protein) and Secondary (for reporter) Antibody
What is an Enzyme-Linked Immunosorbent Assay (ELISA) used to find?
If a specific antibody (protein) binds to a specific antigen or determine the concentration of an antigen in a sample
What does the Sanger method find?
Find the sequence of a particular DNA strand
