Chromatin Flashcards
Each different cell in the body is distinguished by the _____ transcribed in that cell.
genes
______ in transcriptional activity are a major factor in most disease including cancer.
Changes
_____ that alter the normal transcriptional activity of the cell or that alter the RNA processing of specific transcripts are primary causes of genetic disease.
Mutations
Each human cell contains approximately how many feet of DNA?
6 feet (2 meters)
By weight, there is about ____ as much protein as DNA in chromatin.
twice
Approximately how much of all the protein in chromatin is provided by the five histone proteins?
approximately half
*the other half corresponds to additional structural and regulatory proteins
How many different histone proteins are there? What are they named?
5 histone proteins:
- H1
- H2A
- H2B
- H3
- H4
What is the fundamental repeating module in chromatin?
nucleosome
Are histones acidic or basic proteins?
highly basic (positively charged proteins)
*because of their basic charge, they interact strongly with the negatively charged phosphodiester backbone of DNA
What are the “beads on a string” in chromatin?
nucleosomes
What is a nucleosome?
the combined structure of protein and associated DNA
*each bead is a nucleosome
What is the nucleosome core composed of?
- a complex of 8 histone proteins (sometimes called octamer core)
- two copies each of H2A, H2B, H3, H4
- this forms a disk-like structure about 10nm in diameter
What is wrapped around the outside of the histone core?
- DNA (approx. 146 nucleotides of DNA are in direct contact with the histone core)
- about 200 nucleotides per repeating unit of histone core/nucleosome
What does the wrapping of DNA around the octamer core do for the chromosome?
it reduces the length approximately 5-fold
What holds the DNA in place on the histone octamer core?
histone H1 lies on the outside and holds the DNA in place
An entire nucleosome unit is how many base pairs long?
200 bp
Nucleosome modules are wrapped up into a more complex spiral. What is the diameter of this spiral? What is it called?
30 nm
solenoid
Wrapping the nucleosomes into the 30 nm fiber (solenoids) shortens DNA by how much?
it shortens DNA a further 8 times
*i.e. a total of 40x shorter than the linear DNA molecule, because already 5 times shorter from wrapping around histone core
After wrapping of nucleosomes into loops of 30nm in diameter solenoids, these fibers further loop leading to what?
reduction in length of chromatin
-30nm fibers are curled up into whole chromosome fiber
What is the Scaffold structure?
a central structure to which loops of chromatin are attached to
*molecules of chromosomes loop out from Scaffold structure
Scaffold is made of what?
scaffold proteins
By what process is RNA synthesized?
transcription
What does transcription do?
transcription copies the genetic information contained in the primary sequence of DNA into a disposable RNA molecule
Where is RNA polymerase instructed to commence transcription?
at specific sites in the DNA sequence, regulated by proteins that assemble at the promoter region of the gene
The RNA polymerase moves along the DNA and the _____ re-forms as the RNA polymerase passes.
duplex
What does RNA polymerase do?
separates the DNA duplex and then uses complementary base pairing to make a copy of one strand of the DNA molecule
Can multiple RNA polymerases act on a single strand of DNA?
yes - important for genes that are transcribed at very high rates
After the new RNA transcript is approximately 25 nucleotides long, what happens to the 5’ end?
the 5’ end is modified by the addition of an unusual methylated nucleotide, 7-methylguanosine (the ‘cap’)
The 5’ prime end of a new RNA molecule has what?
a triphosphate group
*at first exposed
The 7-methylguanosine (cap) is only found where?
at the 5’ end of a newly synthesized RNA
only found by cap-binding protein
The cap structure contains an unusual linkage between what?
between the 5’ carbon groups of two different ribose sugars (one of cap and one of 5’ end) - sometimes called an ‘inverted’ cap
Where are the enzymes that carry out the capping process?
associated with the machinery of the RNA polymerase complex
What is the enzyme that adds the guanosine (to the cap) called?
guanyl transferase
*methyl group is added by another enzyme
What are the enzymes that are carried along with machinery of RNA polymerase for capping RNA?
capping factors, splicing factors, polyadenylation factors
What region on the RNA polymerase acts as a binding site for many RNA processing enzymes?
the repeating 7 amino acid sequence that is highly modified by phosphorylation
What are the three functions of the mRNA cap structure?
- binds a protein complex that is important for translation
- allows mRNAs to be distinguished from other RNAs without caps
- protects the 5’ end of the transcript against degradation
What is the first step necessary in mRNA degradation?
removal of the 5’ cap
exons
the coding regions of most genes
expressed regions
introns
intervening regions
* copied into the primary RNA transcript exactly like the coding regions
RNA splicing
intron sequences are removed from the primary RNA transcript by this process
*occurs only in the nucleus
What are the two steps of removal of the intron sequences?
- the branch point nucleotide (A) attacks the 5’ splice junction (GU) and precisely cleaves the phosphodiester backbone at this point
- the 3’OH of the first exon sequence then reacts with the 5’ end of the next exon, to precisely excise the intron. The excised material is called the lariat. the lariat structure is rapidly degraded.
The excised material from the RNA sequence is called what?
the lariat, which is rapidly degraded after excision
Despite limited sequence information being present in the primary transcript to direct splicing to the correct locations, what happens that is quite bizarre?
splicing occurs remarkably accurately
What are the sequences that are absolutely conserved (as in kept consistent from strand to strand) in the introns of RNA sequences for splicing?
- GU (at the extreme 5’ end of the intron)
- AG (at the extreme 3’ end)
- the A of the lariat branch junction
fill in
fill in
What are splicing reactions mediated by?
protein/RNA particles called snRNPs (Small Nuclear Ribonucleoprotein Particles)
What are Small Nuclear Ribonucleoprotein Particles (snRNPs) made of?
a small molecule of RNA (less than 200 nucleotides) together with 6 or 7 additional proteins
What are snRNPs assembled into?
they are assembled into larger complexes called spliceosome
What is the spliceosome?
- a dynamic structure, its composition varies during the splicing process
- a complex of snRNPs
- components of the splicing machinery are associated with RNA polymerase as it synthesizes the primary transcript
Where do snRNPs bind and what do they do?
bind to the 5’ end of the splice junction and the A (branch point of the intron)
bring the two regions of RNA together - leading to splicing!
Alternative splicing
different exons may be included or excluded in different tissues or under different physiological conditions
How does alternative splicing work?
Specific proteins bind to the primary RNA transcript and act to block access to certain splice junctions. Mechanisms regulating alternative splicing are currently being actively researched.
It is estimated that there are more than 80,000 different proteins expressed in humans, but there are only about 22-23,000 human genes. What is this difference in numbers mostly due to?
alternative splicing
What genetic disease results from mutation of a splice junction of the intron in the human alpha 2-globin gene?
thalassemia
*A five base deletion changed the GU at the start of the intron, to a GC. Because of this splice site mutation, the a2 globin primary transcript is not spliced correctly and is degraded, resulting in the absence of a2 globin protein.
Systemic Lupus Erythematosus (SLE)
- major symptoms, skin lesions, joint pain, anemia
- SLE is an autoimmune disease
- Patients with SLE make antibodies against snRNPs
- this inhibits correct splicing of multiple genes
Where are approximately 10% of all human disease-causing mutations located?
splice sites
What do mutations at splice sites lead to?
- incorrect splicing of the transcript which may make a mutant protein
- an unstable transcript that is degraded
Transcription continues past the site that will ultimately be the 3’ end of the mRNA (often 100s to 1000s of bases). Correct formation of the 3’ end of the mRNA involves what?
precise cleavage of the primary transcript
After cleavage, what happens to the 3’ end of the mRNA?
the 3’end is modified by the addition of a long tail of adenosine residues - the poly-A tail
What enzyme adds the poly-A tail?
poly-A polymerase
What is poly-A tail bound by?
protein called poly-A binding protein
- this happens after poly-A tail is added by poly-A polymerase
- this protects the 3’ end of the mRNA from degradation by ribonucleases
What is the purpose of the poly-A binding protein?
it protects the 3’end of the mRNA from degradation by ribonucleases
What is the purpose of the poly-A tail?
- increases efficiency of translation
- increases efficiency of transport of the mRNA from the nucleus
- protects the 3’ end of the transcript from degradation
There are sequences defining the site of 3’ cleavage. What is the most important of these signals?
the poly-A addition signal
5’-AAUAAA-3’
located 10-30 bases upstream of the poly-A tail
* a protein binds to this sequence and specifies the cleavage site
If you do not properly cleave the excess nucleotides after the 3’tail end (failure of correct 3’ end formation), what disease is caused?
Beta-thalassemia
What is beta-thalassemia?
- mutation of the T to a C in the middle of the AATAAA sequence in the human beta-globin gene.
- CPSF cannot bind, cleavage does not occur and the transcript is degraded
DNA in the mammalian cell is complexed with a range of different proteins to form chromatin. What is one of the primary functions of this protein association?
to shorten the chromosomal DNA so that it can be packaged within the nucleus
In the mitotic (condensed) chromosome, the DNA length has been reduced a total of about how much?
total of about 10,000x
What are the sequences defining the site of 3’ cleavage?
Analysis of many mRNAs showed the presence of the sequence 5’-AAUAAA-3’ within approximately 10-30 bases of the poly-A tail. This is often called the poly-A addition signal.
In addition to the AAUAAA, additional sequences located
downstream of the cleavage site are also required. These are very poorly defined but usually include a string of U or GU nucleotides. Both the AAUAAA and the downstream sequence are bound by RNA processing proteins associated with the RNA polymerase complex.
After the cleavage event, poly-A polymerase is recruited to add the tail.
