DNA replication, transcription, translation Flashcards
Nucleoside
pentose+base
Nukleotide
phosphate+pentose+base
Nucleic acid
DNA: sugar phosphate backbone and bases
Bond bw. bases
Hydrogen bonds (2 bw. A&T, 3 bw. G&C)
End of the strand
- 3´end of the strand: free 3-OH-gr. of D-ribose
- 5´end of the strand: free 5-P-gr.
Bond be. sugar and P
Esterbond
Deoxyribonucleic acid
DNA. Long polymer of nucleotides as units, w. a backbone of sugar and phosphate.
What part of DNA encodes info.?
Sequence of the 4 bases
What is transcription?
Reading genetic information using genetic code, by copying stretches of DNA into RNA.
What is translation?
Gene expression. The process where mRNA is read and translated into a string of aa.
What is DNA replication?
duplication of chromosomes before the cells divide
Within chromosomes?
chromatin proteins such as histones
Watson-Crick model
conformation of double helix – tertiary structure
Chromosomes in prokaryotes
single circular chromosomal DNA
Plasmid
small circular extrachromosomal DNA
Chromosomes in eukaryotes
many nuclear chromosomes (extrachromosomal DNA: in mitochondria)
Types of proteins in chromosome
histone (alkaline proteins) and non-histone(regulatory and enzyme proteins)
Histone proteins
core 2x (H2A, H2B, H3, H4) and (H1) to stabilize the core histone octamer
Nucleosome
basic unit of eukaryotic DNA. Regularly repeating unit of chromatin
Primary structure of DNA
sequence (order and nr.) of nucleotides
Secondary structure of DNA
base-pairing (H-bonding bw. bases) and the double helix
Tertiary structure of DNA
conformation of double helix (B, A, Z conformation)
Quaternary structure of DNA
- Prokaryotes: one circular chromosome w. superhelices
- Eukaryotes: nuclear chromosomes, containing regularly repeating units of chromosomal nucleoproteins=nucleosomes.
Tm
Define the temp. of which half of the DNA is melted. Melts into ss (single stranded) DNA. Varies on G+C content. If G+C content is more than T+A content, Tm is more. Reverse is A+T rich DNA
Conformation (tertiary structure)
- B form: double helix - turn every 0,34 nm -> 10 base pairs
- A form: structure change, in sol. w. higher salt conc. or alcohol added - 2,3 nm->11 base pairs
- Z form: zigzag - 4,6 nm->12 base pairs
Type of conformation in prokaryotic DNA
Contains superhelix (double double helix). Positive (overtwisting) or negative (untwisting).
Topological isomers
-Coiled DNA
Type I topoisomerase
cuts one strand of ds DNA, relax the strand and then reanneal the strands. No ATP is needed.
Type II topoisomerase
cuts both strands of DNA helix, results in undwinding, neg. superhelix. ATP is needed. E.g. DNA gyrase (derived from Escherichia coli, bacterial).
Topoisomerase inhibitors
antimicrobial or antitumour agents
What encodes one aa?
3 bases (triplet=code) of DNA
Nr. of possibilities to prod. triplets?
64 possibilities to produce triplets from the 4 different nucleotides (A, G, C, T).
Are genes continious or discontinuous, and what does it consist of?
Discontinuous, consist of coding exons and non-coding introns.
Synthesis of DNA
Semiconservative replication
- DNA helicase
- Replication fork
- DNA polymerase, 3´to 5´end
- Complementary nucleotides, 5´to 3´end
- Leading strand (5‘ to 3‘)
- Lagging strand (3‘ to 5‘)
- Okazaki fragments, DNA ligase
Diection of the new strand synt. in prokaryotes?
5´to 3´
Which side of the DNA synthesis is continious/discontinous?
On the leading strand the synthesis of the new strand is continuous, on the lagging strand is discontinuous.
Initiation of replication
- Begins in the replication origo, recognized by DNA A protein.
- Primer-start molec.
- Synt. by primase
- Helicase
- All of these proteins form replisome.
Primer
Short DNA segment, start molecule, has a free 3´end.
Helicase
(in DNA B and C proteins) binds to ss. regions of DNA to prevent premature annealing of DNA strands.
Elongation of replication
- Primase synt. short RNA oligonucleotides copied from DNA.
- DNA polymerase III elongates RNA primers with new DNA.
- DNA polymerase I removes RNA at 5´end and fills gap
- DNA ligase connects adjacent fragments.
DNA polymerase isoenzymes in prokaryotes
- DNA polymerase I and II: removal of primer, filling of the gaps. Repair of DNA.
- DNA polymerase III: Synthesis of DNA. Found in enzyme complex, molecular machine (replisome) carries out replication.
DNA polymerase at eukaryotes
- DNA polymerase alpha: synthesis of nuclear DNA
- DNA polymerase beta: Removal of primer, filling of the gaps. Repair of DNA.
- DNA polymerase gamma: synthesis of mitochondrial DNA.
Telomers
In Eukaryotic DNA replication, to the end of the chromosomes protecting telomeres (repeating DNA sequences) can be synt.
Mutation
Change in base sequence of the gene results in mutant protein
Types of mutation
•Point mutation: -Substitution, such as transition: alternative pyrimidines (C, T) or purines (A, G) or transversion: purine pyrimidine (C/T, A/G). -Same sense (silent) -Missense -Nonsense •Insertion, such as duplication •Deletion Consequence: frameshift •Multisite mutation: gene, chromosome, genom
Functional difference bw. DNA and RNA
DNA to DNA: replication, DNA to RNA: transcription) of genetic info
Which type of RNA has the largest molecular mass of all types?
Messenger RNA (mRNA)
Which type of RNA transports covalently bound (activated) aa. to the protein synt.
Transfer RNA (tRNA)
What is codes and codons?
Triplets of DNA=codes, triplets of mRNA=codons
How many possible variations is there in gene expression?
64 possible variations -> 3 stop codons + 61 variations for 20 proteinogenic aa. (1 start codon – AUG – Met)
Needed for transcription
•DNA template/non-coding/antisense strand
•Building stones for the synt. of RNA (ribonucleoside-triphosphate molecules, NTPs)
•Enzyme
-Prokaryote: DNA dependent RNA polymerase
-Eukaryote: DNA dependent RNA polymerase I, II and III
Types of DNA dependent RNA polymerase in eukaryote
- I: synt. of rRNA
- II: synt. of mRNA and snRNA
- III: synt. of tRNA (+rRNA and snRNA)
What is the promotor region in prokaryotes?
pribnow-box and CAP-cAMP binding site
What is the promotor region in eukaryotes?
GC-box and TATA-box (core promotor)
Steps in transcription
- Initiation
- Elongation
- Termination
Needed for initiation in transcription in prokaryotes?
- RNA polymerase must bind sigma-factor
* cAMP-CAP site must be engaged by cAMP-CAP complex
Needed for elongation in transcription in prokaryotes?
- Transcription bubble
- NTP -> NMP + PPin
- RNA polymerase builds NMPs in the chain of mRNA being synt.
Needed for termination in transcription in prokaryotes?
•p(rho)-factor dependent -loop is formed in mRNA -RNA polymerase -ATP •p(rho)-factor independent (rarely) -loop
is mRNA polycistronic/monocistronic?
Polycistronic
Where can Shine-Dalgarno sequences be found?
bw. genes in Prokaryotic mRNA = ribosome binding site
Where can Untranslated regions (UTR) be found?
on 5´and 3´ends of Prokaryotic mRNA
Who has posttranscriptional modification?
Eukaryotes
Regulation of prokaryotic transcription
- Operon model
- CAP-cAMP binding site on promotor region
Transcription in prokaryotes happens only if?
- CAP-cAMP complex is bound to CAP-cAMP binding site of promotor
- Repressor protein is not bound on the operator region
Types of operons
- Lac-operon: transcription of genes of enzymes needed for breakdown of lactose
- Lac-operon: promotor region, operator region, structural genes: Z-gene (beta-galactosidase), Y-gene (permease), a-gene (transacetylase)
- Tryptophan operon: transcription of genes of enzymes synt. tryptophan
What is an operon?
distinct segment of DNA, a complete set of different regions
Needed for initiation in transcription in eukaryotes?
- Helicase subunit
- RNA polymerase II
- Protein kinase subunit
Needed for elongation in transcription in eukaryotes?
•NTP -> NMP + PPin
-RNA polymerase II
What does RNA polymerase II do?
Build NMPs
When is termination in eukaryotes?
At cleavage sequence, after UTR
What does helicase?
Uncoils DNA during initiation in transcription of eukaryotes, in the region of START site, by using 1 ATP
What does Protein kinase?
During initiation in transcription of eukaryotes, hydrolyses 1 ATP for the phosphorylation of RNA polymerase II
Steps in posttranscriptional modification?
- 5´capping
- Polyadenylation (polyA-tailing)
- Splicing
What is 5´capping?
7-methyl-GTP cap is built on the 5´end of mRNA (protection from enzymatic cleavage+transport)
What is Polyadenylation (polyA-tailing)?
100-200 pcs of AMP is built on the 3´end of mRNA by polyA polymerase -> formation of polyA-tail (protection from enzymatic cleavage+transport)
What is splicing?
removal of introns and ligation of exons to form mature mRNA
Where does splicing happen?
Nucleus
Types of splicing?
- Alternative splicing
- Trans-splicing
What is mature eukaryotic mRNA composed of?
7-methyl-GTP cap, UTR- exons (ligated), UTR, polyA-tail
How does regulation of transcription happen in eukaryotes?
- Altering chromatin structure (epigenetic regulation)
- Histone acetylation
- DNA-methylation
Transcription factors
Proteins bound to the promoter region
•Activator protein -> incr. gene expression -> gene enhancing
•Repressor protein -> decr. gene expression -> gene silencing
Synthesis of rRNA
- Pre-rRNA -> rRNA by methylation
- DNA dependent RNA polymerase I (III)
Synthesis of tRNA
- Pre-tRNA -> tRNA by binding 3´CCA sequence (aa. binding site)
- DNA dependent RNA polymerase III
What does gene expression involve?
Transcription+translation.
DNA –(transcription)-> RNA –(translation)-> protein
How does gene expression happen in the prokaryotic cell?
transcription and translation happens at the same time
Loops in translation
- 1st loop - DHU (dehydrouridine) loop: binds aminoacyl-tRNA synthetases.
- 2nd loop - anticodon loop containing the complimentary triplet codon to that on the mRNA.
- 3rd loop - pseudouridine loop (T, pseudouridine, C): binds tRNA to the ribosome
Where will the aa. be bound to during translation?
The acceptor stem-3´end of the tRNA molecule
What recognize which codon to bring an aa during translation?
tRNA, by anticodon on its mRNA-binding end that is complementary to the codon on the mRNA
What is responsible for recharging tRNA with another aa. during translation?
aminoacyl-tRNA synthetases (ARS)
Amount of swedbeg units in ribosomes of prokaryotes?
30 and 50S
Amount of swedbeg units in ribosomes of eukaryotes?
40 and 60S
Type of ribosome units?
Small (30 or 40S) and large (50 or 60S) subunits
Start codon for prot. synt.?
AUG=codes for the aa. methionine. This aa. is used to start synt. of both prokaryotic and eukaryotic proteins.
Needed in initiation in translation of prokaryotes?
Initiation complex: IF2 (initiation factor-2)-GTP-formylmethionyl-tRNA
Needed in initiation in translation of eukaryotes?
Pre-initiation complex: IF2 (initiation factor-2)-GTP-methionyl-tRNA
What is Kozaks-scanning?
Start codon scanning. Need 1 ATP/base.
Elongation in prokaryotes?
- An aminoacyl-tRNA binds in A site. The anticodon loop binds to the codon of mRNA.
- Peptide bond is formed by peptidyl transferase, so Met will be bound to aminoacyl tRNA on the A site. Non-charged tRNA will be bound in the ribosomal E site, and then leaves the ribosome.
- Translocation occurs
Required for aminoacyl-tRNA to bind in A site?
GTP and EF-1 alpha
Required for translocation during elongation?
GTP, EF-2 and translocase
Difference bw. eu- and prokaryotic elongation:
Instead of EF-1 alpha, EF-Tu exists
- In prokaryotes transcription and translation on the same polypeptide chain happens at the same time
- In prokaryotes poly(ribo)some is produced
What is wobbling?
The third base of a codon is less important for id. the correct anticodon than the first two
What are the stop codons?
UAA, UGA and UAG
What is termination faciliated by?
By binding of protein called protein releasing factor (RPF) to the stop codon. RPFs recognize stop codons.
When does termination occur?
when one of three special codons called stop codons appears in the A site of the ribosome
Regulation of translation
- If the complementarity of mRNA and small subunit of rRNA is big, the intensity of protein synt. is also big.
- If an aa. is coded by a preferated codon of mRNA, the appropriate tRNA will be found quicker, so the intensity of prot. synt. is also bigger.
- micRNA complementer to 5´end of mRNA can inhibit competitively binding of mRNA to the small subunit, so prot. synt. will decr.
Posttranslation modifications
1.Splitting a peptide or an aa.
•Proinsulin becomes mature insulin
•Zymogens prod. active enzymes
•Met or fMet removal
2.Phosphorylation/dephosphorylation of OH in Ser, Thr, Tyr
3.Hydroxylation: OH-Lys and OH-Pro in collagen
4.Glycosylation of Ser-OH (in membrane)
5.Redox Rs: disulphide bond form.
6.Acetylation: histone proteins
•Transport of the modificated, final forms of proteins: bound to signal sequences
