1
Q
What is the difference in the pentose sugars in RNA compared to DNA?
A
- RNA: ribose sugar containing 2’ hydroxyl (OH) group
- DNA: 2’ deoxyribose is missing the OH group, only has a proton (H)
2
Q
Which is more reactive, DNA or RNA?
A
RNA is more reactive than DNA
3
Q
What mechanism allows RNA to fold and form a secondary structure?
A
hydrogen bonding between complementary bases on the same strand
4
Q
How are genes usually organized in prokaryotes?
A
in prokaryotes genes are usually organized as a single (continuous) coding unit
5
Q
How are genes usually organized in eukaryotes?
A
genes in eukaryotes are usually split or interrupted: exons are protein coding segments, introns are intervening (non-coding) segments
6
Q
What does the unique organization of genes in prokaryotes allow in terms of transcription?
A
in prokaryotes transcription (making of RNA) occurs simultaneously (coupled) with process of translation (if RNA destined to encode a protein)
7
Q
Describe the organizational features of a gene in prokaryotes?
A
- starts with region that regulates transcription, then region that regulates translation followed by the coding region and the signals for termination of transcription
8
Q
Describe the initiation and termination codon on the primary transcript:
A
- AUG initiation codon shortly after 5’ end
- UAA termination codon shortly before 3’ end
9
Q
Describe the simultaneous transcription, translation and mRNA degradation in prokaryotes
A
- RNA transcripts are made by several RNA polymerase molecules transcribing along the DNA
- located on the RNA molecules are many ribosomes that translate the RNA into protein (called polyribosomes) simultaneously with production of RNA chains by RNA polymerase
- after sufficient molecules of protein are made, the RNA polymerase disassembles and RNA is often degraded
10
Q
How does the process of transcribing and translating DNA occur in eukaryotes with their interrupted genes?
A
- RNA transcripts are made and processed in the nucleus and then must be transported to the cytoplasm for translation
- one step in nucleus, one step in cytoplasm, therefore physically and temporally separated steps
11
Q
What must happen in eukaryotes in order to produce mRNA from DNA?
A
- a primary transcript is made and includes the introns as well as the exons
- then the introns must be removed
- now we have mRNA
- mRNA must be transported to the cytoplasm
- in the cytoplasm the mRNA, which consists only of coding regions, can be translated into a polypeptide
12
Q
Are most types of RNA translated into protein?
A
No, most RNAs remain as RNA and function that way either structurally or catalytically
- mRNA is the only RNA that gets translated into protein
13
Q
What is mRNA?
A
Messenger RNA = an intermediate that carries genetic information from DNA to ribosomes
14
Q
What is tRNA?
A
Transfer RNA = has structural and catalytic components of ribosomes
15
Q
What is snRNA or snoRNA?
A
= small nuclear RNA = spliceosomes and rRNA, tRNA modification
16
Q
What are the functions of miRNA, siRNA, and Crispr RNA?
A
they are all micro RNAs (very short) that block the expression of complementary mRNAs
17
Q
What are long noncoding RNAs?
A
= long RNAs that regulate gene transcription
18
Q
How are the DNA strands used during transcription?
A
- only one of the strands is used as a template
19
Q
Which DNA strand is used as the template for transcription?
A
= 3’ to 5’
- means that RNA is made by RNA polymerase in the 5’ to 3’ direction using the 3’ to 5’ DNA strand as the template
20
Q
In which direction is RNA synthesized?
A
5’ to 3’ - new nucleotides added to 3’ OH group of growing RNA
it is complementary and anti-parallel to the DNA template strand, meaning that it is synthesized from the 3’ to 5’ DNA strand
21
Q
If RNA is transcribed 5’ to 3’ what does this mean for gene transcription on the 5’ to 3’ DNA strand?
A
- genes can be located on either strand but transcription will always occur in 5’ to 3’ direction
- therefore they will be transcribed in a different direction on the 5’ to 3’ strand compared to the 3’ to 5’ strand of DNA, i.e. transcription can use either strand as 3’ to 5’ template but 5’ to 3’ will be transcribed left to right while 3’ to 5’ is transcribed right to left
22
Q
Which elements are required chemically for transcription?
A
- DNA template
- 4 ribonucleoside triphosphates (rNTPs): A, U, C, G
- DNA dependant RNA polymerase
( - reaction buffer such as found in cell)
23
Q
What occurs chemically during transcription?
A
- an RNA molecule that contains a number of ribonucleotides (RNAn) can be extended by addition of a rNTP complementary to the 3’ to 5’ DNA tempalte
- nucelophilic attack of 3’ OH of ribonucleotide in the growing chain on alpha phosphate of incoming rNTP, forms phosphodiester bond and releases PPI (pyrophosphate)
- this extends the RNA chain by one base
24
Q
What are the general features of RNA synthesis?
A
- it is similar to DNA synthesis except in the following
- precursors are ribonucleoside triphosphates (rNTPs)
- only one strand of DNA is used as the template (the 3’ to 5’ strand)
- RNA chains can be initiated de novo (no primer required)
- the 5’ to 3’ RNA molecule = complementary to DNA template 3’ to 5’ (DNA anti-sense strand) and identical to DNA non-template known as DNA sense (except T changed for U)
- RNA synthesis is catalyzed by RNA polymerases and always proceeds in 5’ to 3’ direction
25
Who articulated the central dogma first?
Francis Crick in 1958
26
Which type of RNA also has a tertiary structure?
tRNA
27
What are the components of the generalized structure of a PROKARYOTIC gene?
- the two DNA strands (3' to 5' strand is template for transcription)
- DNA sequence that signal where RNA polymerase will begin process of transcription = promoter region
- the actual start site of transcription
- the continuous coding region of prokaryotic gene
- DNA sequence signalling end of transcription (terminator sequence)
28
Are the promoter and terminator region included in the RNA transcript?
- the terminator region is included
| - the promoter region precedes the transcription start site and is therefore not included in transcript
29
Is the promoter upstream or downstream of the coding region?
upstream
30
Is the terminator upstream or downstream of the coding region?
downstream
31
Does RNA polymerase need a primer to begin synthesis?
No, RNA polymerase simply recognizes and binds to DNA sequences in promoter region and initiates transcription
32
What are the main steps in prokaryotic transcription?
1. Initiation
2. Elongation
3. Termination
33
What happens during initiation in prokaryotic transcription?
RNA polymerase binds, unwinds, and joins first 2 nucleotides
| - initiation does NOT require a primer
34
What occurs during elongation in prokaryotic transcription?
= complementary nucleotides continue to be added during the elongation process
- localized DNA unwinding ahead of RNA polymerase generates a transcription bubble
- transcription bubble moves with the RNA polymerase and the unwound DNA rewinds behind it
35
What happens during termination in prokaryotic transcription?
= transcription stops when RNA polymerase reaches the terminator region of the gene
- newly-synthesized RNA together with RNA polymerase are released
36
Describe prokaryotic RNA polymerase:
- it is a multi-subunit complex
| - the RNA polymerase cor can transcribe any segment of DNA but there are also specialized subunits called sigma subunit
37
Describe the core of prokaryotic RNA polymerase:
- transcribes any DNA sequence, not gene specific
- alpha subunit involved in assembly of tetrameric core
- beta subunit contains ribonucleoside triphosphate (rNTP) binding site
- beta-prime subunit contains DNA template binding region
- omega subunit helps to stabilize the tetrameric core *alpha, beta, and beta-prime
38
Describe the holoenzyme of prokaryotic RNA polymerase
- alpha, beta, beta-prime, omega, and sigma
- the structure of the complete RNA polymerase, sigma structure is specific for transcribing genes
- sigma subunit binds to RNA polymerase tetrameric core and assists in correct initiation of transcription specifically at promoter region of prokaryotic gene
- sigma gives RNA polymerase specificity for the gene
39
What cellular environment does RNA polymerase require to properly function?
- must include divalent cation Mg2+
40
What is required for recognition of gene promoter region during initiation of prokaryotic transcription?
the intact RNA polymerase holoenzyme = tetrameric core plus sigma factor
- sigma factor recognizes and binds to -35 element in promoter region, which properly positions RNA polymerase to begin transcription
41
What are two important sequence elements in the promoter region of prokaryotic transcription?
- the -35 element to which the sigma factor binds
- the -10 element, which due to its very A/T rich content is prone to unwinding
- both are located before the transcription start
42
What does sigma factor binding permit in prokaryotic transcription?
- permits transcription of the gene sequences to begin at the adjacent transcription start site
43
What is the Pribnow box?
the -10 sequence like in the E.coli promoter region
44
What does the -35 region sequence look like?
5'-TTGACA 3'
45
What does the -10 sequence look like?
5' TATAAT 3'
46
What separates the -35 from the -10 sequence?
16-19 base pairs of "spacer" DNA
47
Where is the transcription start site located relative to the -10/Pribnow box sequence?
about 5-9 base pairs after Pribnow box, it is a Purine (A or G) on the non-template strand, so a pyrimidine (T or C) on the template strand (3' to 5') about 5-9 pairs after the end of the -10 sequence (5' TATAAT 3')
48
Describe the 5' end of RNA and its significance
5' end is usually a purine and is called the +1 position
- this occurs because RNA polymerase simply recognizes the promoter sequence and starts transcribing at the T or C 5-9 bp after -10 sequence without requiring a primer
49
What does the elongation stage of transcription in prokaryotes involve?
- release of the sigma factor followed by the steady movement of RNA polymerase along the 3' to 5' template strand of the DNA
- involves transcription bubble that accompanies RNA polymerase
- RNA polymerase has built in helicase activity which assists with unwinding duplex DNA ahead of polymerase
- topoisomerases have to move ahead of polymerase to remove any positive supercoils nicking and then re.-ligating DNA
50
Describe the transcription bubble involved in prokaryotic transcription?
- involved in elongation stage
| - a localized region of unwinding that involves approx. 18 base pairs
51
Can RNA polymerase unwind and rewind the DNA helix?
Yes, RNA polymerase has both helix unwinding and rewinding activities
52
How are new rNTPs added to RNA chemically?
- new rNTPs are added via phosphodiester bond formation between 3' OH of preceding ribonucleotide and alpha phosphate of the incoming rNTP with elimination of PPi
53
Describe the common mechanism of transcription termination in bacteria:
- termination is rho-independent
- weak H-bonding at U:A residues allows mRNA release from DNA when RNA polymerase pauses at terminator
- happens after the palindromic sequences allow for the formation of the stem loop or hairpin structure in the RNA
54
What are the palindromic sequences in rho-independent transcription termination?
- palindromic means that on each DNA strand the beginning of the sequence on one side is complementary to the beginning of the sequence on the other side, this means that RNA containing these sequences can hydrogen bond and form a stem-loop or hairpin structure
55
What is the significance of the stem-loop or hairpin structure in rho-independent transcription in prokaryotes?
- during transcription the hairpin structure resides within the catalytic core of the RNA polymerase and causes it to pause transcription
- this allows for newly-synthesized RNA to release from DNA template due to weak hydrogen bonding in the adjacent A:U rich region
- ends the transcription process for that particular RNA polymerase
56
What are Balbiani rings?
- enlarged open regions of DNA seen for instance in the very large chromosomes in the salivary glands of insects, Balbiani rings = aka puffs
i. e. localized unwinding due to gene transcription in these eukaryotes
57
How many RNA polymerases do most eukaryotes have?
3 RNA polymerases
58
What are key facts about eukaryotic RNA polymerase I?
- found in all eukaryotes
| - transcribes large rRNAs
59
What are key facts about eukaryotic RNA polymerase II?
- found in all eukaryotes
| - transcribes pre-mRNA, some snRNAs, snoRNAs, and some miRNAs
60
What are key facts about eukaryotic RNA polymerase III?
- found in all eukaryotes
| - transcribes tRNAs, small rRNAs, some snRNAs, and some miRNAs
61
What are key facts about eukaryotic RNA polymerase IV?
- only found in plants
| - transcribes some siRNAs
62
What are key facts about eukaryotic RNA polymerase V?
- only found in plants
| - transcribes RNA molecules that take part in heterochromatin formation
63
How many RNA polymerases exist in prokaryotes?
we only know of 1
64
How do the RNA polymerases 1-3 work?
they all have specialized accessory proteins that direct specific transcription for each polymerase, so they all recognize specific promoters in the genes, so Pol I, Pol II, and Pol III are only recruited to their specific promoters
65
How do eukaryotic and prokaryotic promoters compare?
Eukaryotic promoters are more complex
66
Describe the RNA polymerase II promoter
- consists of a core promoter and a regulatory promoter that aid in positioning transcription proteins and RNA polymerase II to begin transcription
67
What is the core promoter in RNA polymerase II transcription?
= -35 sequence, rich in G:C (CGCC) bp and the TATA box (A:T rich: TATAAA) around position -25 upstream from transcription start site
68
What do the regulatory regions do in RNA polymerase II transcription?
- located in core promoter, assist in positioning of RNA polymerase II at beginning of gene and facilitate transcription
69
What does initiation in transcription in eukaryotes with RNA polymerase II involve?
- ordered assembly of transcription factors of RNA polymerase II: TFII, A, B, C, D, E, F, and H
- TFII(TFID) assembles first by binding to the -25 sequence (TATA box) in core promoter followed by binding of other TFs and RNA polymerase II = pre-initiation complex (PIC)
- interaction b/n PIC and regulatory proteins aids in starting transcription
70
What is the "mediator" in complex RNA polymerase II transcription?
- a multi-subunit complex that permits interactions with other activator proteins bound to upstream/downstream regulatory regions or enhancer sequences
71
What happens during the elongation stage of prokaryotic DNA transcription?
- many of the general transcription factors remain at promoter providing for quick reinitiation with a new Pol. II
- an approx 8 nucleotide transcription bubble is generated by RNA:DNA binding
- this together with DNA unwinding ensures that the free RNA 3'-OH terminus is available for new rNTP addition
72
What is alpha amanitin?
- a potent toxin from death cap mushroom that inhibits RNA polymerase II initiation and elongation by blocking the polymerase catalytic core
- can lead to death within 10 days
73
Describe termination of transcription in eukaryotes
- polymerase transcribes past the coding sequence of most genes, then pauses
- long pre-mRNA is cleaved in special sequence found at the end of the gene by Rat1
- transcription terminates when Rat1 reaches RNA polymerase
74
Describe how Rat1 works
- has both endo- and exo-nuclease activities and can both cleave the pre-mRNA and digest the remaining unuseable part of the transcript which stops the transcription by pol II and causes everything to dissociate (5' to 3' degradation)
- leaves functional part of the pre-mRNA available for further processing
75
What does it mean that the sequence of genes in prokaryotes is "co-linear" with the amino acid sequence of the protein?
- with colinearity the number of nucleotides in the gene is proportional to the number of amino acids in the protein
- therefore, coding sequence of gene is co-linear with mRNA and polypeptide
76
What is the Shine-Delgarno sequence?
5' UAAGGAGGU 3' = Shine-Delgarno located on mRNA!
- involved in initiation of translation in Prokaryotes
- it is followed by the ATG start codon, next is protein-coding region, then translation stop codon
77
What is RNA splicing?
- the process by which introns (intervening, non-coding segments in eukaryotic pre-mRNA) are removed from pre-mRNA
- intron removal makes mRNA, which is a continuous coding segment that can be made into a polypeptide
78
Where do transcription and RNA processing steps occur in eukaryotes?
in the nucleus of the cell
79
What are the 3 main pre-mRNA processing steps in eukaryotes?
1. Addition of 7-methyl guanosine cap
2. Addition of PolyA tail
3. Removal of introns
- important for export of mRNA, protecting it from degradation and facilitating translation inititation
80
Explain the processing step of 7-MG addition in pre-mRNA processing:
- addition of 7-methyl Guanosine (7-MG) cap
- linked to pre-mRNA by a unique linkage between 5' phosphate of the 7-MG and 5' phosphate of the first ribonucleotide in the RNA (5' to 5' linkage)
- occurs early in elongation process
81
Explain the addition step of a PolyA tail in pre-mRNA processing
- pre-mRNA is cleaved and then a long string of A residues is added by Poly A polymerase
- aka polyadenylation
- occurs after Rat1 endonuclease has digested pre-RNA
- follows after the recognition sequence 5' AAUAAA 3', here it is cleaved by RAT1 and then poly A polymerase adds a string of approx 200 A residues at cleaved end
82
Explain the removal of introns in pre-mRNA processing
- introns in pre-mRNA are removed by a specialized process called RNA splicing - must be precse to properly fuse the 3' end of one exon to the 5' end of the next exon
83
How does the eukaryotic cell ensure that introns are properly removed?
- every intron has two conserved sequences that are required for its precise removal:
1. 5' to 3' splice sequences containing junction sequences GU and AG respectively
2. Intron branch point: a conserved A residue: within central portion of intron is a conserved A residue
- these sequences are used by spliceosome to correctly remove each intron and ligate 3' and 5' ends of each exon
84
What is the spliceosome?
- an RNA/protein complex that contains 5 small nuclear RNAs (snRNAs) designated U1, U2, U4, U5, and U6
- snRNAs associate with about 40 small proteins to form small nuclear ribonucleoproteins (snRNPs)
- snRNPs, U1, U2, U4/U6 and U5 assemble to form a complete spliceosome
85
What is the mechanism the spliceosome uses to remove introns from eukaryotic pre-mRNA?
- snRNP U1 binds to 5' splice site and snRNP U2 binds to branch site (snRNP assembly)
- complete spliceosome assembles and cleaves at 5' splice site
- the 5' end of intron is joined to A in the branch site to form a lariat structure and U1 and U4 are released = 3' splice site cleaved
- exons are joined when 3' splice site cleaved and 5' end of exon 3 is joined to 3' end of exon 1, lariat shaped intron is released along with snRPS U2, U5, and U6
86
What does lariat formation involve?
a unique linkage between the 5' phosphate of the G and the 2' OH of the A
87
How are Group I and II introns removed from rRNA precursors?
autocatalytically by rxn of the RNA molecule itself
- Group I and II introns are located in genes of bacteria, bacteriophages, eukaryotes, archaea, and eukaryotic organelles
- i.e. a "self-splicing" process
88
How are introns of tRNA precursors excised?
by precise endonucleolytic cleavage and ligation rxns
| - tRNA genes are located in bacteria, archaea, and eukaryotes and are excised through enzymatic rxn
89
Which introns are removed by spliceosomes?
nuclear pre-mRNA introns
| - located in protein/encoding genes in nucleus of eukaryotes
90
How can one gene make many proteins?
- through the presence of introns which allows for alternative modes of splicing and 3' end processing that can generate protein diversity
- in humans alternative processing occurs in more than 90% of genes
- i.e. many different mRNAs can be produced from a single gene through specialized treatment of pre-mRNA
91
What is 'alternate splicing of introns'?
- selective removal of introns by the spliceosome that produces several mRNAs containing different exons (therefore different but related proteins)
- i.e. two different mRNAs can be made from single pre-mRNA of the gene by alternative splicing
92
Explain processing of pre-mRNA that involves multiple 3' cleavage sites
- in pr-/mRNA this can result in production of mRNAs with different 3' ends which can also result in different but related proteins
- can splice at different 3' sites which reslts in products of different lengths
MBG 2040
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