Biochemistry of Nucleic Acids Flashcards
what is the central dogma?
DNA > RNA > Protein
difference between ribose and deoxyribose?
ribose has 2 OH groups, deoxyribose only has 1
how are carbons numbered in nucleotides/nucleosides?
from amino end to phosphate end
DNA building blocks vs RNA building blocks?
DNA = dATP, dCTP, dGTP, dTTP RNA = ATP, CTP, GTP. UTP
how does polymerisation occur in DNA?
phosphodiester bond is formed between a free 3’ OH group on the above nucleotide and a 5’ triphosphate on ATP, leaving 2 diphosphate
consumes 2 high energy bonds
give an example of a nucleotide analogue used as a drug and describe how it works
ZDV/AZT/Retrovir (analogue of thymidine)
incorporated into growing viral DNA but lacks 3’ OH group so chain elongation is terminated
how many bond between A/T and C/G?
A-T = double bond C-G = triple bond
replication is conservative, true or false?
false
only semi conservative
what catalyses DNA replication?
DNA polymerase
can only add to existing nucleic acid and require an RNA primer to start replication
in which direction is DNA replicated?
bidirectional
always 5’ to 3’
building blocks of DNA replication?
dATP, dTTP, dCTP, dGTP
what synthesises an RNA primer?
primase
what is the exonuclease activity of DNA polymerase?
moves 3’ to 5’ removing incorrect nucleotides
improves error rate
most abundant RNA?
rRNA (80%)
forms ribosomes
tRNA = (15%)
mRNA = (5%)
describe structure and function of tRNA
clover leaf structure when flattened
specific amino acid is attached to 3’ end (dependant on anti-codon sequence)
what are the 3 types of eukaryotic RNA and how can they be distinguished?
Pol I, ii and iii (Pol ii makes all mRNA)
by sensitivity to toxins like alpha amanitin
what is TBP?
TATA box Binding Protein
recognises TATA box (present in promotor region)
part of TFIID
introduces kink into DNA providing landing platform for further transcription factors and RNA polymerase
what is TFIID?
general transcription factor required for all Pol ii transcribed genes
how is transcription initiated?
needs additional general transcription factors
Pol ii and TFIIF extend transcript on their own
TFIID remains at promotor, a new initiation complex can now assemble
allows transcription at low, basal rates
how is the transcribed strand elongated?
transcription bubble moves along DNA from 5’ to 3’ (DNA unwound in front of polymerase and rewound behind it)
how is transcription terminated?
new RNA makes a stem loop structure (followed by stretch of U’s)
specific enzyme cleaves the finished RNA
RNA released and polymerase dissociates
how is transcription regulated?
needs specific transcription factors (DNA binding proteins with DNA binding domain and transcriptional activation domain)
bind to specific DNA sequences in vicinity of promoter (enancers)
describe coordinated gene expression
a stress (e.g hormone stimuli, cellular stress etc) activates transcription of a regulatory protein through a stress sensitive transcription factor
binding of the regulatory protein to the stress regulatory element (SRE) stimulates transcription of genes A, B and C
Genes A, B and C produce different proteins which participate in the stress response
give 2 examples of coordinated gene expression
steroid receptors (on binding, ligand (steroid) moves to nucleus and binds to DNA at steroid response elements (SRE) glucocorticoid receptors (free steroids enter target cells via diffusion, bind to inactive steroid receptor in cytoplasm, activates receptor, translocates to nucleus, binds to response elements as homodimer, coordinated regulation of set of genes)
when does splicing take place?
after transcription, before translation
what happens at the 5’ and 3’ ends of mRNA?
Poly(A) tail (AAAAA) added at 3’ end
modified GTP “cap” added at 5’ end
what is the difference between transcription/translation in prokaryotes and eukaryotes?
compartmentalised in eukaryotes
- transcription in nucleus
- translation in cytosol
occurs anywhere in prokaryotes
degenerate vs unambiguous?
degenerate = amino acid has more than one codon unambiguous = each codon has only one amino acid (or a stop)
start vs stop codons?
start = AUG stop = UAA, UAG, UGA
what are the 3 different reading frames of RNA (e.g if there are an extra 2 bases which cant complete a codon)?
2 bases at the right end
1 base either side
2 bases at the left end
what are the components of translation?
amino acids tRNAs Aminoacyl-tRNA synthetases Specific protein factors for - initiation of protein synthesis - elongation - termination ATP and GTP (energy) Ribosomes mRNA
what does aminoacyl tRNA synthetase do?
attaches amino acid to its tRNA via covalent bond
at least 1 per amino acid
how many rRNA molecules are contained within each ribosome?
4
3 in bacteria
what are the components of a ribosome?
4 rRNA molecules
proteins
which has bigger ribosomes, prokaryotes or eukaryotes?
eukaryotes
3 tRNA binding sites of ribosomes?
Exit
Peptidyl
Aminoacyl
initiation of translation?
needs initiation factors (IFs)
energy from GTP hydrolysis
small ribosomal subunit binds to 5’ end of mRNA
moves along mRNA until start codon met
initiator tRNA with UAC anticodon base pairs with start (carries methionine)
large subunit joins assembly and initiator tRNA located in P site
elongation of translated protein?
elongation factor (EF-1alpha) brings next aminoacyl-tRNA to A site (anticodon-codon) GTP hydrolysed, EF released from tRNA second elongation factor (EF-1betagamma) regenerates EF1alpha
what catalyses the peptide bond formation between amino acids and where does this occur?
peptidyl transferase
occurs in P and A sites
how does the ribosome move along the mRNA?
elongation factor EF-2 moves it along by 1 triplet at a time
what is the pathway a tRNA takes in a ribosome of a growing peptide?
moves from A to P and then the empty tRNA moves to the E site where it can exit and become reloaded with an amino acid
where are the A, P and E sites located on a ribosome?
A = furthest towards 3' end E = furthest towards 5' end P = in the middle
how does termination occur?
when A site encounters a stop codon (no aminoacyl tRNA pairs with a stop codon)
release factor RF binds stop codon (GTP hydrolysis)
finished protein cleaved off tRNA
rRNA, mRNA and tRNA dissociate from each other
what is a polysome?
mRNA with several ribosomes attached
point mutation?
change in single DNA base
missense mutation?
causes change in amino acid sequence
can change protein function (sickle cell anaemia)
Nonsense mutation?
creates new termination codon
changes length of protein due to premature stop of translation
silent mutation?
no change in amino acid sequence
due to degeneracy of genetic code
no effect on protein function
frameshift mutation?
addition or deletion of single base (or 2)
changes reading frame of translation into protein
types of chromosomal mutations? how are they different?
deletions duplications inversions translocations affect larger portions of genome
what are the 3 options for a finished protein?
targeting (moving to final cellular destination, depends on amino acid sequence)
modification (adding further functional groups)
degradation (unwanted or damaged proteins have to be removed)
what do free ribosomes do and where are they found?
make proteins for - cytosol - nucleus - mitochondria - translocated post-translationally found in cytosol
what do bound ribosomes do and where are they found?
make proteins for - plasma membrane - ER - Golgi apparatus - secretion - translocated co-translationally found on rough ER
what are the 2 destinations for newly synthesised proteins?
to organelles/cytosol
to rough ER
give 4 examples of post translational modifications that can occur in the ER
glycosylation (adding/processing carbohydrates in ER an Golgi)
forming disulphide bonds
folding/multiunit assembly
proteolytic cleavage (in ER, Golgi and secretory vesicles)
what results from misfolding of alpha1-antitrypsin in the ER?
hereditary emphysema
give a clinical example of protein targeting
I-cell disease (recessive)
proteins destined for lysosomes not properly sorted and end up secreted from the cell
lysosomes cant digest material and become clogged
death before age 8
