topic 7 - Nucleic Acid (HL) Flashcards
How did Hershey and Chase provide evidence that DNA is the genetic material of the cell and not protein?
Known that viruses insert their genetic material into cells
radioactive viruses grown –> allowed to infect a bacterium (E. colu)
- Viruses grown in 35S had radioactive proteins (no DNA) but did NOT transfer this radioactivity to bacterium (remained in supernatant)
- Viruses grown in 32P had radioactive DNA (no protein) and DID transfer this radioactivity to infected bacterium (found in pellet)
virus & bacteria separated via centrifugation. Showed the above results
This demonstrated that DNA, not protein, was the genetic material because DNA was transferred to the bacteria
Who used X-ray diffraction to elucidate DNA structure?
Franklin and Wilkins
What is X-ray Diffraction?
- X-rays will diffract when targeted at crystallised DNA molecules
- The scattering pattern created can be used to determine structure
From the scattering patter created by X-Ray Diffraction, what are the properties that can be deduced?
- Composition: DNA is a double-stranded molecule
- Orientation: The bases face inwards and the phosphates face out
- Shape: DNA forms a double helix (10 bases per twist)
True or False?
In eukaryotes, DNA associates with eight histone proteins to form a nucleosome
TRUE
How does nucleosomes help to supercoil DNA?
- Makes DNA compact (better storage)
- Prevents DNA damage (less exposed)
- Assists in cell division (more mobility)
- Involved in transcriptional regulation
What are the regions of DNA that do not code for protein?
Think: STING
- Satellite DNA (tandem repeats)
- Telomeres (chromosome ends)
- Introns (non-coding sequences)
- Non-coding RNA genes
- Gene regulatory sequences
What are used in DNA profiling?
Tandem repeats
Describe how DNA –> Chromosome
DNA is bound with histone proteins to form nucleosomes that are then linked together to form strings of chromatosomes
These coil to form solenoids, which condense into 30 nm fibres, before being compressed and folded into chromatin
Chromatin will then supercoil during cell division to form chromosomes that are visible (when stained) under microscope
What is the role of helicase in DNA replication?
- Helicase separates the DNA strands to form a replication fork (breaks the hydrogen bonds between complementary base pairs)
- Single stranded binding proteins prevent strands re-annealing
What is the role of DNA Gyrase in DNA replication?
- DNA gyrase reduces the torsional strain created by helicase
- It prevents the DNA from supercoiling as it is being unwound
What is the role of DNA primase in DNA replication?
• DNA primase generates a short RNA primer on each strand
• PrimersprovideaninitiationpointforDNApolymeraseIII
(DNA pol III can only add nucleotides to 3’-end of a primer)
What is the role of DNA Polymerase III in DNA replication?
- Free nucleotides (dNTPs) line up opposite complementary bases
- DNA polymerase III covalently joins free nucleotides together
What is the role of Okazaki Fragments in DNA replication?
- DNA strands are antiparallel, so replication occurs bidirectionally (replication always occurs in a 5’ → 3’ direction on each strand)
- Synthesisiscontinuousontheleadingstrand(towardsfork) and is discontinuous on the lagging strand (away from fork)
- Discontinuous segments are called Okazaki fragments
What is the role of DNA Polymerase I in DNA replication?
• DNA pol I removes RNA primers and replaces them with DNA
What is the role of DNA ligase in DNA replication?
• DNA ligase covalently joins the Okazaki fragments together
Briefly describe the DNA replication process
- origins in DNA (eukaryotes have multiple, prokaryotes only have ONE)
- helicase unwinds DNA
2a. single-stranded binding (SSB) proteins keep DNA separated
2b. topiosomerase prevents supercoiling - Primase makes RNA primers
- DNA polymerase III builds the new strand in ONLY 5’ –> 3’ direction; proof reading ability prevents mistakes
- DNA polymerase I removes primer
- ligase seals Okazaki fragments on lagging strand tgth
- new strand
What is DNA sequencing?
Sequencing is a technique by which the nucleotide base order of a DNA sequence is elucidated (typically via Sanger method)
Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing (use of chain-terminating dideoxynucleotides)
Briefly describe the mechanism of DNA sequencing. What is the Sanger method?
Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group needed to form covalent bonds (they terminate replication)
SANGER METHOD:
Four PCR mixtures are prepared – each with stocks of normal bases and one dideoxynucleotide (ddA, ddT, ddG, ddC)
Whenever the dideoxynucleotide is randomly incorporated, the DNA sequence is terminated at that base position
Because a complete PCR cycle generates millions of sequences, every base position is likely to have been terminated
These sequences are separated by gel electrophoresis to determine base sequence
(according to ascending sequence length)
Automated machines can determine the sequence quickly if fluorescent labeling of the dideoxynucleotides has occurred
True or False?
A gene is a sequence of DNA which is transcribed into RNA • Most genes encode proteins, but some do not (e.g. tRNA)
True
What are the 3 main sections of a gene sequence?
- Promoter (transcription initiation site)
- Coding sequence (the region transcribed)
- Terminator (transcription termination site)
True or False
As DNA is double stranded, only one strand is transcribed
• The antisense strand is not transcribed into RNA
• The sense strand is transcribed into RNA
FALSE
As DNA is double stranded, only one strand is transcribed
• The antisense strand is transcribed into RNA
• The sense strand is not transcribed into RNA
True of False?
Gene expression is regulated by proteins (transcription factors) that bind to specific sequences associated with a promoter
True
True or False?
Transcription Factors:
Activators bind enhancer sites (⬇︎︎ rate of transcription)
False
• Activators bind enhancer sites (⬆︎︎ rate of transcription)
True of False?
Transcription Factors
• Repressors bind silencer sites (⬇︎︎ rate of transcription)
TRUE
Gene expression is regulated by proteins that bind to specific base sequences in DNA.
What are regulatory proteins? What is their purpose?
The presence of regulatory proteins may be tissue-specific or may be influenced by chemical signals (e.g. hormones)
proteins bind to DNA sequences outside of the promoter and interact with the transcription factors
Type of Regulatory Proteins:
What are activator proteins?
bind to enhancer sites and increase the rate of transcription (by mediating complex formation)
Result: high levels of transcription
Type of Regulatory proteins:
What are repressor proteins?
bind to silencer sequences and decrease the rate of transcription (by preventing complex formation)
Result: low levels of transcription
What are Basal transcription factors?
gene transcribed at moderate levels
Result: normal levels of transcription
How do nucleosomes help regulate transcription in eukaryotes?
Histones proteins have protruding tails that determine
how tightly the DNA is packaged within nucleosome
How does modifications to the protruding tails of histone proteins alter DNA packaging?
Acetylation makes DNA less tightly packed
Methylation makes DNA more tightly packed
Cells package DNA differently according to genetic needs. How are active genes packaged?
Active genes remain unpackaged as euchromatin
Cells package DNA differently according to genetic needs. How are inactive genes packaged?
Inactive genes are tightly packed as heterochromatin
What is DNA methylation? How does this affect transcription?
DNA can also be directly methylated to change expression patterns of genes over time in response to external stimuli
• Increased methylation = decreased transcription
True or False.
DNA methylation patterns influenced by heritability but is not genetically pre-determined (identical twins may have different DNA methylation patterns)
True
DNA methylation patterns differ b/w clones (twins) and change over time
Eukaryotic cells modify RNA after transcription has occurred. Modifications must occur to produce mature mRNA. Thus, non-coding regions within genes are removed. What is this process called?
Splicing
What are introns?
Introns are non-coding regions in genes
THINK: intruding
What are exons?
• Exons are the coding regions of genes
THINK: expressing
What is alternative splicing?
Exons can be selectively removed to form different proteins from the same gene
What is an examples of non-coding DNA? What is its functions?
Example: promoter
The non-coding sequence responsible for the initiation of transcription
The core promoter is typically located immediately upstream of the gene’s coding sequence
The promoter functions as a binding site for RNA polymerase (the enzyme responsible for transcription)
The binding of RNA polymerase to the promoter is mediated and controlled by an array of transcription factors in eukaryotes
These transcription factors bind to either proximal control elements (near the promoter) or distal control elements (at a distance)
what is capping in transcription?
Capping involves the addition of a methyl group to the 5’-end of the transcribed RNA
The methylated cap provides protection against degradation by exonucleases
It also allows the transcript to be recognised by the cell’s translational machinery (e.g. nuclear export proteins and ribosome)
What is polyadenylation in transcription?
Polyadenylation describes the addition of a long chain of adenine nucleotides (a poly-A tail) to the 3’-end of the transcript
The poly-A tail improves the stability of the RNA transcript and facilitates its export from the nucleus
The environment of a cell and of an organism has an impact on gene expression
True of False?
True
Chemical signals within the cell can trigger changes in levels of regulatory proteins or transcription factors in response to stimuli
This allows gene expression to change in response to alterations in intracellular and extracellular conditions
Also organisms responding to environmental changes:
Hydrangeas change colour depending on the pH of the soil (acidic soil = blue flower ; alkaline soil = pink flower)
True of False?
Ribosomes are the site of polypeptide synthesis. They are composed of ribosomal RNA and protein
TRUE
Polypeptide synthesis IS translation
What are the 2 subunits that ribosomes consist of?
- Small subunit contains an mRNA binding site
* Large subunit contains three tRNA binding sites (E, P, A
Fill in the Blank.
Multiple ribosomes can translate a single mRNA sequence simultaneously (these are collectively called a __________)
Multiple ribosomes can translate a single mRNA sequence simultaneously (these are collectively called a POLYSOME)
What is transfer RNA (i.e. tRNA)?
Transfer RNA (tRNA) carries amino acids to the ribosome • Amino acids are attached by tRNA-activating enzymes
What are the two steps that tRNA-activating enzymes function.
- The enzyme joins ATP to an amino acid (‘charging’)
* ‘Charged’ amino acid is linked to tRNA (AMP is released)
Fill in the Blank
The purpose of ‘charging’ the amino acid is to __________________
The purpose of ‘charging’ the amino acid is to
ANS: create a high energy bond that can be be used during translation
The purpose of ‘charging’ the amino acid is to create a high energy bond that can be be used during translation. What does ribosomes use this energy for?
Ribosomes use this energy to synthesise peptide bonds
Fill in the Blank
Each tRNA-activating enzyme is specific to a particular amino acid, but may bind multiple tRNA. Due to __________
Degeneracy
True or False
In prokaryotes, the absence of a nuclear membrane allows translation to occur immediately after transcription.
True
In eukaryotes, translation will occur at one of two locations. What are these two locations? Describe each one
• Free ribosomes (cytosolic) synthesise intracellular proteins
• Bound ribosomes (rER) synthesise proteins destined for
secretion from the cell or for use in lysosomes
True or False.
Translation can occur immediately after transcription in prokaryotes due to the absence of a nuclear membrane
TRUE
What is a polysome?
a group of two or more ribosomes translating an mRNA sequence simultaneously
appear as beads on a string (each ‘bead’ represents a ribosome ; the ‘string’ is the mRNA strand)
What is the function of free ribosomes?
Free ribosomes synthesise proteins for use primarily within the cell
What is the function of bound ribosomes?
Bound ribosomes synthesise proteins primarily for secretion or for use in lysosomes
