Biochem DNA --> Protein Flashcards
What is a nucleoside?
A nucleotide base and a sugar (ribose in RNA, deoxyribose in DNA)
eg. adenosine, cytidine, guanosine, thymidine and uridine
What is a neucleotide?
A nucleotide base, a sugar and a phosphate group
What are purines?
Neucleotide bases A and G. Double ring bases
What are pyrimidines?
Nucleotide bases T + C + U. Single ring bases
What are the DNA building blocks?
dATP, dCTP, dGTP and dTTP
deoxyadenosine triphosphate
What are the RNA building blocks?
ATP, CTP, GTP and UTP
adensine triphosphate
What is polymerisation of DNA building blocks?
When a phosphodiester bond is formed between a free 3’OH group and a 5’triphosphate. Consumes 2 high energy bonds as the pyrophosphate ion bonds are hydrolysed.
Nucleic acids have direction. Which end are nucleotides added too and which direction is the sequence read?
New nucleotides are added to free 3’ ends
Sequence is read 5’-3’
AZT (ZDV) is an antireteroviral drug. How does it work?
Analogue of thymidine and is incorporated into viral DNA but it has no 3’ OH group meaning their is no chain elongation.
Works as a viral reverse transcriptase and has a high affinity for ZDV than human DNA polymerases
The DNA double helix is antiparalel. What does this mean?
2 nuceotide strands- one 5’-3’ and one 3’-5’.
How many hydrogen bonds between AT and CG?
AT = 2 CG = 3
When is DNA replicated?
Before cell division
How is DNA replicated?
Semi conservative replication
Which enzyme unzips DNA and also prevents rewinding?
DNA helicase
Which enzyme catalyses the addition of nucleotide to a single strand of DNA in replication?
DNA polymerase
Can DNA polymerase begin a new single strand of DNA?
What is needed to initiate replication?
No- it can only add to existing strands.
RNA primer is needed to start DNA replication
How many origins of replication in eukaryotes and prokaryotes?
Eukaryotes = many Prokaryotes = one
Why is having many origins of replication beneficial for eukaryotes?
Ensures replication can be finished in a reasonable length of time.
Replication starts simultaneously at several points on the genome and is bidirectional forming a DNA bubble with 4 sites for DNA polymerase
DNA replication is discontinuous. What is meant by this?
Neucleotides can only be added to a 3’ end.
Generates a leading strand- always has a free 3’ end and a lagging strand which is replicated in short segments
How is the lagging strand replicated in DNA replication?
OKAZAKI fragments. RNA primers are needed and the gaps between okazaki fragments are joined by DNA ligases
What synthesises RNA primers?
DNA primase
What joins the gaps between okazaki fragments?
DNA ligase
How frequently does a mutation in DNA replication occur and how is this reduced?
10^4-10^5 base pairs
DNA polymerase has 3’–>5’ endonuclease activity and removes incorrect nucleotides by back tracking
Reduces error rate to 10^9-10^10 base pairs.
Other repair systems = nucleotide incision repair
What are the 3 main classes of RNA?
1) Ribosomal RNA (rRNA) 80%
2) Transfer RNA (tRNA) 15%
3) Messenger RNA (mRNA) 5%
Can RNA contain local stretches of intramolecular base pairing forming stem loops?
Yes as its usually single stranded
What is the function of rRNA?
Combine with protein to from ribosomes. Stable
What is the function of tRNA?
Carries the amino acid to be transported to a protein. Stable.
What is the function of mRNA?
Genetic information for protein synthesis- very long
What are the features of tRNA?
Anticodon of 3 nucleotides
Specific amino acid attached to the 3’ end
Distinct 3D structure but 2D looks like a clover leaf
How is RNA produced?
Produced by RNA polymerases.
Eukaryokes have 3 = Pol I, Pol II, Pol III
Pol II synthesises all mRNA
Prokaryotes only have one.
Which RNA polymerase produces all the mRNA in eukaryotes?
Pol II
What are the 5 steps of transcription?
1) RNA polymerase binding: detection of promotors on DNA and requires transcriptional factors
2) DNA chain separation: local unwinding of DNA
3) Transcription initiation: selection of the first RNA nucleotide
4) Elongation: Addition of further nucleotides to the RNA chain
5) Termination: release of the finished RNA
Transcription: Explain RNA polymerase binding?
RNA Pol II binds to a specific promoter which is determined by the following:
1) A TATA box is present at -25
2) TATA box binding protein (TBP) recognises the TATA box and is part of TFIID, a general transcriptional factor
3) TBP induces a kink in DNA which determines the transcriptional start and direction (transcription begins at nuceotide +1. No nuceotide 0)
4) TFIID provides a platform for other transcriptional factors and Pol II to bind.
What is the role of transcriptional factors?
To switch the transcription of the genes on and off
Transcription: After DNA is unwound and the double stands separated by DNA helicase, transcription is initiated. How does this happen?
1) Requires more general transcriptional factors with a precise order of assembly
a) Pol II and TFIIF extend the transcript
b) TFIID remains at the promoter allowing transcription at basal rates
Transcription: How is the RNA elongated after initiation?
1) A transcription bubble moves in one direction and DNA is unwound in front of Pol II
2) RNA is synthesised in a 5’–>3’ direction
Transcription: The RNA strand is identical to which, the template or the coding strand?
The coding strand. It is complementary to the template strand
Transcription: How is transcription terminated?
Newly synthesised RNA mays a stem loop structure follwed by a stretch of Us
A specific enzyme cleave the RNA which is released and RNA polymerase dissociates
How is transcription regulated?
DNA binding proteins (specific transcriptional factors) Contain 2 functional domains which can be separated:
1) DNA binding domain (regulatory)
2) Transcriptional activation domain
ENHANCERS- bind to specific DNA sequences in the vicinity of a promoter to regulate transcription
How can external stimuli (stress) activate transcription for specific proteins?
Using a specific transcriptional factor (regulatory protein) which binds to the stress response element which stimulates transcription of many genes => proteins to relieve stress
What are steroid receptors?
Family of transcriptional factors that are inactive in the cytoplasm but when bound to a steroid they move to the nucleus and bind to DNA at steroid response elements
Activates steroid response genes
What are exons?
Coding segments of RNA
What are introns?
Non coding sements of RNA (1-50 introns per gene)
What is pre mRNA and how is it transformed to mRNA?
mRNA from transcription which still contains introns. Transformed to mRNA by splicing
Where does splicing occur?
Nucleus
What is the significance of the splice variant protein?
Allows you to skip exons so one gene can code for many proteins
How are the ends of mRNA processed during splicing?
Addition of a poly A tail to 3’ end
Addition of a G cap at 5’ end to prevent unraveling
* stabalises the mRNA
Where does translation occur?
Cytoplasm
How many amino acids in the genetic code and what are the characteristics of the genetic code?
20 amino acids
1) Degenerate- many amino acids have more than one codon
2) Unambiguous: each codon codes for only one amino acid or a stop codon
3) Universal: most organisms use the same genetic code
What are the stop codons?
UAG, UAA, UGA
What is the start codon?
AUG (methionine)
Translation: What are reading frames?
Each RNA molecule can be translated in 3 different reading frames depending on where transcription starts
What componenents are needed for translation?
Amino acids tRNA Ribosomes mRNA ATP/GTP for energy Aminoacyl- tRNA synthases Specific set of protein factors
Translation: what are the specific set of protein factors needed for?
1) initiation of protein synthesis
2) Elongation of the peptide chain and translocation
3) Termination
Translation: What are aminoacyl- tRNA synthases?
Proteins which bind amino acids to the corresponding tRNA molocule at the 3’ end. Specific and unique to each amino acid
Translation: what provides the energy for the covelent bond to form between tRNA and an amino acid and how is the amino acid activated to then be bound to tRNA?
ATP is needed for the covelent bond
ATP is also needed along with an enzyme to form an AMP amino acid which is activated
How many RNA molecules in a ribosome?
4 and protein components
S is a unit of sedimentation (linked to density). Which S are found in eukaryotic ribosomes?
60S and 40S subunits forming an 80S ribosome
What are the 3 tRNA binding sites on ribosomes between the large and small subunits?
E = exit P = Peptide A = aminoacyl
How is translation initiated?
1) Initiation factors and GTP is hydrolysed for energy
2) Small ribosomal subunit binds to the 5’ end of mRNA and moves along mRNA to AUG
3) initiator tRNA with UAC anticodon base pairs with AUG
4) Large suunit joins assembly and initiator tRNA is located in the P site
How is the peptide elongated in translation from when initiator tRNA is in the P site?
1) Elongation factor (EF-1 alpha) brings the next aminoacyl tRNA to the A site => anticodon pairs with codon
2) GTP is hydrolysed and EF is released from tRNA
3) Second EF (EF- beta gamma) regenerates EF 1 alpha to pick up the next aminoacyl tRNA
4) Peptide bond formation and translocation fallows
Translation (elongation): How is a peptide bond formed and how does translocation occur?
1) Peptidyl transferase catalyses peptide bond formation between amino acids in the P and A sites.
2) The peptide now sits in the A site
3) EF 2 moves the ribosome along mRNA by one triplet so empty tRNA moves to the E site and tRNA with peptide moves to the P site and site A is free for the next aminoacyl tRNA
When is translation terminated?
Occurs when the A site encounters a stop codon as no aminoacyl tRNA pairs with a stop codon
How is translation terminated?
Release factor binds to stop codon and GTP is hydrolysed. The fished protein is cleaved off tRNA. The components dissociate.
What is a polysome?
Created when a single mRNA is translated by many ribosomes simultaneously
What is a point mutation?
Change in a single base in DNA
What is a missense mutation?
change in amino acid sequence
WHat is a nonsense mutation?
Creates a premature stop codon and chages the length of a protein
What is a silent mutation?
No change in amino acids and no effect on protein function
What is a frame shift mutation?
Addition or deletion of 1 or more bases which changes the reading frame
What is a chromosomal mutation?
Mutation effecting a larger portion of the genome- deletions, duplication, inversions and translocation
What happens to finished proteins?
1) Targeting- moving a protein to its cellular destination
2) Modification- addition of further functional chemical groups
3) Degradation- unwanted or damaged proteins are removed
What do free ribosomes make proteins for?
Cytosol, nucleus and mitochondria
Translocated post translationally
What do bound ribosomes make proteins for?
Plasma membrane, ER, Golgi and secretion
Translocated co translationally
Post translation modification: what is glycosylation?
addition and processing of carbohydrates in the ER and golgi
Post translation modification: where are disulphide bonds formed?
ER
Post translation modification: where does the folding and assembly of multisubunit proteins take place?
ER, golgi and secretory vesicles
Post translation modification: what is phosphorylation?
Adding phosphate groups to activate the protein
Post translation modification: why is proteolysis important?
Cleaving the polypeptide allowing the fragments to fold into different shapes
