Maintaining the Cell and Genome Flashcards
What is a genome?
Complete set of genetic information in an organism
Provides all the information an organism requires to function
Genome is stored in long molecules of DNA: chromosomes
In eukaryotic cells, the genome is contained within the nucleus
How is DNA stored/organised in a cell?
DNA packaged with histones (positive charged) – form nucleosome structure – nucleosome are the functional unit of chromatin – fold into a tightly coiled structure - ultimately condenses into chromosomes
What are the building blocks that make up DNA? What are they composed of?
The basic building block of DNA is the nucleotide
- 5-carbon sugar
- Phosphate moiety
- Nitrogenous base - nitrogen containing ring
Things to note…
1. Sugar-phosphate backbone gives the DNA strand polarity - 5’ carbon of one sugar binds to the 3’ carbon of the next via a phosphodiester bond
2. Four different types of bases - A, T, C and G
What are the two groups of nitrogenous bases?
Note:
- Purines are bulkier as they have a double ring structure
What type of bonding holds two DNA strands together?
A-T – two hydorgen bonds
C-G – three hydrogen bonds
What is the difference between a nucleotide and a nucleoside?
Do DNA strands have directionality?
YEAH BUDDY
How are nucleosomes formed?
First and most fundamental ‘level’ of chromatin packing is the nucleosome:
Histones + DNA – nucleosome 200 Bp – come together to form ‘beads on a string’ chromatin
What are the subunits that make up histones? What other important features do histones have?
Histones – DNA-binding proteins (small positively charged proteins).
Two each of: H2A, H2B, H3 & H4 - forming an octamer
Histone tails are significant – susceptible to a large number of covalent modifications
After nucleosome formation, what are the following levels of DNA condensation/packaging?
- First order packing – beads on a strings – 7 fold reduction in length
- Chromtain fibres - looping/organising histones
- Chromtain fibres undergo further looping
- Formation of mitotic chromosome
How many chromosome, autosomes and sex chromosomes do we have?
23 chromosomes - Haploid Cell
46 chromosomes - Diploid cells = 3 billion nucleotide (base) pairs
22 pairs of autosomes
1 pair sex chromosomes
What is meant by the idea that DNA replication is semi-conservative?
Due to WC base pairing - each strand acts as template for synthesis of a new complementary strand - Base pairing enables DNA replication
Hence, after replication the new DNA strand will carry a parent and a new daughter strand - hence, DNA replication is semi-conserved.
Where does DNA replication begin? Where does DNA replication take place? What are these sites called?
DNA synthesis begins at replication origins
Replication origin – sites where DNA replication starts – need to seperate strands and create a replication bubble (break open hydrogen bonds) - creates single stranded templates ready for DNA synthesis
Initiator proteins recognize replication origin and unwind DNA at these sites
Multiple origin sites can be active at one time
DNA synthesis occurs at replication forks
Two forks form at each origin
Replication proceeds bidirectionally, unzipping the DNA strands as it goes
Most important enzyme in DNA replication is DNA polymerase
In what direction is DNA synthesized?
DNA is synthesised in the 5’ to 3’ direction
What big problem is encountered in DNA replication?
At a replication fork, the two newly synthesised DNA strands are of opposite polarities
This creates a problem for DNA polymerase, which can only synthesise DNA in a 5’-3’ direction
How does DNA replication of the lagging strand take place?
Solution – synthesizes fragments on the lagging strand in a 5’ to 3’ (work in the opposite direction to the replication fork) forming Okazaki fragments
These can then be ligated together
This is known as discontinuous synthesis
Why are primers needed for DNA replication? What enzyme is responsible for laying primers down?
Problem - DNA polymerase can only continue an existing strand, not initiate new ones - hence, this is why primers are needed
An RNA polymerase known as primase makes RNA primer first (~10 bases long) - DNA polymerase can then extend the RNA chain
Note
- Only one RNA primer needed for leading strand, but lagging strand has continuous requirement
- RNA later removed by nuclease activity
Does DNA polymerase proof-read its own work?
Yes, DNA polymerase proofreads its own work
Replication must be very, very accurate (although mutations must arise at some frequency, or evolution couldn’t occur)
Consequences of errors can be fatal
Allowing for proofreading, DNA polymerase makes around 1 error in 10^7 bases
What is the overall accuracy of DNA replication? What mechanism exists to correct these mistakes?
Overall accuracy of DNA replication is ~1 error in 10^9 bases
Mutations can be passed on to offspring if they take place in germline cells but can also be problematic in somatic cells
DNA mismatch repair
Machinery recognizes error and new daughter strand – chews back and allows DNA polymerase to jump back in to repair
What are causes of DNA damage?
What is an example of DNA damage associated with the base thymine?
Exposure to ultraviolet light can form thymine dimers
If they can’t be repaired - results in a condition called Xeroderma pigmentosum (Genetic defect) - results in severe skin lesions, including skin cancer
What is the principle behind DNA repair pathways?
What are the differences between mitochondrial and nuclear DNA?
What is the central dogma of molecular biology?
What are the differences between DNA and RNA?
Extra
DNA – mainly used as information storage
RNA – plays a variety of roles – catalytic, make proteins, etc.
Which carbon in the sugar moiety of DNA and RNA is different?
Carbon 2
RNA - Carbon 2 is hydroxylated
DNA - Carbon 2 is not hydroxylated
What is DNA transcription?
Transcription - Process that produces an RNA copy of the coding strand of DNA (except that it contains U, not T)
Note - the non-coding strand is used as a template to form a coding strand
What enzyme is responsible for transcription?
Transcription is carried out by RNA polymerase
mRNA is made by RNA polymerase II - Makes an RNA copy of one DNA strand
Recognizes start site - unwinds DNA - proceeds along DNA while transcribing new strand - new strand is released from complex - stop site reach - complex is released and DNA can re-anneal
Requires
1. DNA template
2. Activated precursors (nucleoside triphosphates - ATP, GTP, UTP and CTP)
3. Does not require a primer
4. Synthesises in 5’- 3’ direction
Note
- Error rate is higher – mistakes have less severe consequences than DNA mutations
Describe what happens in the RNA polymerase complex during transcription.
- DNA enters complex
- DNA is unwound
- Exposing the non-doing/template strand to incoming rNTPs
- rNTPs are added to the growing RNA chain (short DNA-RNA hybrid)
- RNA released from DNA
- DNA is allowed to reanneal
How do we get tissue/cell specific expression of genes?
Genes may be either on (expressed) or off in a given cell - not all genes are expressed in all tissues at all times
Genes may be expressed in a:
1. Tissue-specific pattern
2. Developmentally regulated pattern
3. Combination of these
4. “on” all the time, in all tissues (constitutive)
- e.g. housekeeping genes
How?
- Transcription factors interact with the 5’ promoter and the enhancer sequences to regulate expression (increase or decrease)
What is the transcription factor responsible for myosin gene expression?
Presence of transcription factors MyoD will dictate whether the myosin gene is transcribed or not
How can a single ‘gene’ give rise to different protein products?
Expression of a single gene can be controlled at various levels to give different products:
- Alternative promoters - different transcription start sites (TSS) used resulting in different products.
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Alternative splicing - primary transcript can be spliced in different ways, joining different exons together, resulting in different products (not exon fundamental order can’t be changed - they can simply be skipped)
e.g. Example: the alpha-tropomyosin gene can be spliced in many different ways - Alternative polyadenylation
Before exiting the nucleus, what modifications do mRNA transcript undergo?
Before RNA can be exported from the nucleus it must go through RNA processing steps:
- Splicing
- Capping
- Polyadenylation
What is RNA capping? Why is it important?
Modifies the 5’ end of a RNA transcript
The RNA cap includes an atypical nucleotide: a guanine nucleotide that has a methyl group attached to the 5’ end of the RNA in an unusual way
Important for… protecting RNA from degradation, stabilization, stimulates splicing and drives nuclear export
What is polyadenylation? Why is it important?
A polyA tail is a string of adenylate residues added to the 3’ end of an mRNA
Not found on rRNAs or tRNAs - Labels the RNA as mRNA
Transcription proceeds past polyA site, transcript is cleaved, then polyadenylated
What are 5’ and 3’ UTRs? Why are they important?
5’ untranslated (5’UTR) is the region of an mRNA that is found upstream of the translated region
Function of 5’UTRs is mostly unclear, may affect translational control
3’ untranslated (3’UTR) is the region of an mRNA that is found downstream of the translated region
3’UTRs can determine the stability of the mRNA
What are the three major classes of RNA?
What are some examples of non-coding RNAs and their functions?
Extra
1. Circular RNA species - Class of RNA molecules with closed loops, high stability, abundantly expressed in eukaryotic organisms - thought to regulate gene expression and protein translation
What is PCR? What is it used for? What are the steps undertaken in this reaction?
The Polymerase Chain Reaction (PCR) - DNA amplification - can be used for genotype - detecting mutations in patients
Steps
1. Strand Seperation
2. Primer annealing
3. Extension
What happens during the process of translation?
Translating – change in ’code/language’ - change from nucleotides (DNA/RNA) into protein
The RNA code is read in triplets (codon - three bases at a time) - with each triplet codon corresponding to a specific amino acid
What is meant by the genetic code being redundant?
Redundancy in the genetic code - multiple codons coding for a single amino acid - triplets can code for 64 different combinations but there are only 20 amino acids
Note
- Third position in the codon is commonly redundant
- There are also start (AUG) and stop codons (UAA, UGA and UAG)
How are codons read? What are reading frames?
- Code is read in the 5’ to 3’ direction
- Three different reading frames – depends where you start counting the triplets
- Important that translation starts at the correct start position/reading frame
What molecule is responsible for translating/bringing amino acids to the corresponding codon?
tRNAs (transfer RNAs) are key adaptors required for protein synthesis
tRNA has a 3’ end that attaches to a specific amino acid and a anti-codon region that binds to the codons on the mRNA strand - therefore allowing it to bring the corresponding correct amino acid
Note
- Some tRNAs require accurate base-pairing only at 1st two nucleotides
- This ‘wobble’ accounts for finding that many alternative codons for a particular amino acid differ only in their 3rd nucleotide
How is the correct amino acid added to the corresponding tRNA molecule?
tRNA synthetases are needed to transfer the correct amino acid to the corresponding tRNA
Recognizes anti-codon and matches with the correct amino acid – relies on ATP hydrolysis to bind the two together (charging of the tRNA) – forms a high energy bond which is required for the addition of the amino acid to the growing peptide chain
What are ribosomes? What role do they play in translation?
Ribosomes - the factory of translation - very complex
- Made of RNA (rRNAs) and proteins
- Has large & small subunits
- Large subunit – catalyzes the peptide bond formation – covalently linking amino acids
- Small subunit – matches tRNA to mRNA
- Ribosome moves along the mRNA transcript, in a 5’ to 3’ direction
Role - guides translation and catalyzes the polymerisation reaction
What are the three tRNA docking sites on ribosomes?
How does it coordinate this reaction
Three tRNA sites and mRNA binding sites
1. A site – aminoacyl site - tRNA binding
2. P site – Peptidyl site - peptide bond formation/polymerisatrion
3. E Site – exit
Outline the steps that take place in the translation cycle in order to produce a polypeptide chain.
- tRNA for start codon binds to the P site directly (bringing in Methionine)
- tRNA with the matching anti-codon for the codon in the A site binds
- Peptide bond is formed between the amino acid in the P and A site – causes release of the amino acid from it’s tRNA
- Ribosome shifts across - Large subunit translocates, followed by small subunit translocation
- Spent tRNA in the E site leaves
- Vacant A site can be filled by new tRNA molecule
- Process repeats.
How/where is translation started and stopped?
- Translation initiates at a methionine codon (AUG) near 5’ end of mRNA. Therefore, methionine is always the first amino acid added (may be removed later)
- Translation terminates at a stop codon. Three stop codons, UAA, UAG and UGA - Stop codons not recognised by a tRNA, release factor binds instead
Do some antibiotics target prokaryotic translation?
Yes, some antibiotics work by inhibiting prokaryotic protein synthesis
This is possible as there are differences in the structures of prokaryotic and eukaryotic ribosomes
