Lecture 36
Difference between transcription and DNA replication
transcription occurs without a primer because RNA polymerase can initiate the synthesis of a new RNA chain using the template DNA strand. Transcription can occur without a primer, unlike DNA replication
What is promoter escape?
After incorporation of the first 10 nucleotides, the sigma factor must dissociate upon transition from the transcription initiation to transcription elongation, known as promoter escape. The sigma factor will dissociate and the remainder of the RNA polymerase will move down the DNA transcribing mRNA, a stable elongation complex
What is the "open promoter" complex
After the RNA polymerase binds, it unwinds several base pairs from -12 to -1 giving it a stable "open promoter" complex. This consists of RNA polymerase holoenzyme and a promoter that has undergone initial unwinding in preparation for the start of transcription. DNA is bent in this complex so the bases are accessible to be read by the polymerase for transcription. An Mg2+ ion is also present in the catalytic site which chelates negative charges on incoming nucleotide triphosphate.
Considering that RNA transcription is much slower than DNA replication (50 nucleotides added/second versus 500-1000 nucleotides/second), how is it possible that RNA >> DNA in living cells?
Although RNA polymerases are slower than DNA polymerases, there are only a few DNA polymerase molecules expressed in a living cell, whereas there are thousands of RNA polymerase molecules expressed at the same time. RNA polymerases can initiate transcription at many more sites than there are origins of DNA replication, so it makes sense to have more RNA polymerases in the cell.
Why is it important the 5' end of mRNA is capped in eukaryotes
Protects 5' end from exonuclease attack and is important for initiation of translation.
RNA Pol II synthesizes
mRNA
Rho-dependent termination of transcription (less frequent)
occurs when the Rho protein disassociates the RNA polymerase and moves it off the template.
What are the two strands that result from the promoter region being unwound used for
one strand is used as a template and the other will not. the template strand is read by the RNA polymerase to make an mRNA.
Genetic organization of prokaryotic genomes
organized into blocks of functionally related genes, called operons. The entire prokaryotic genome is contained in a single circular DNA molecule. These genes are co-transcribed, meaning one RNA transcript contains coding sequences for more than one protein.
what 3 modifications do eukaryotic mRNA make
- 5' cap - 3' poly(A) tail - splicing of introns
Describe how the 5' end of mRNA is capped
- 5' terminal triphosphate has one phosphate group removed and GMP is added from GTP. This is a 5' to 5' linkage. The G' is then methylated at the 7' N position and 2' OH of the 2nd/3rd nucleotide.
Rho-independent termination mechanism (more frequent)
- GC region slows down the polymerase because of its tight base pairing, making the template hard to unwind. This gives the RNA transcript time to base pair to itself, forming the stem-loop structure and displacing the RNA from the DNA template. The DNA-RNA base pairing is weakened. - Further weakening between the DNA template-RNA transcript occurs when the run of A's are transcribed to give a series of A-U bonds, which are weak. transcript released from template
Large complex TFIID contains?
- TBP: TATA binding protein binds to TATA "box", bends DNA - TFIIB binds to TBP and DNA and recruits polymerase and opens DNA
2 domains of transcription factors?
- They usually have two domains (fragment) that may be in separate parts of the molecule or overlapping; 1, DNA binding domain that binds to the enhancer and 2, transcriptional activation domain
Two types of termination events
- factor independent (rho-independent) - rho dependent termination
Eukaryotic vs prokaryotic initiation of transcription
- for prokaryotes, initiation of transcription does not need any proteins or initiation factors - in eukaryotes, initiation of transcription requires proteins called transcription factors
eukaryotes vs. prokaryotes transcription factors
- in eukaryotes, initiation of transcription requires proteins called transcription factors. instead of a sigma factor, they have transcription factors that recognize promoter regions and can influence transcription - in prokaryotes, initiation of transcription does not need any proteins or initiation factors.
What are the eukaryotic transcription DNA sequence control elements
- initiator sequence near start site (YYANWYY) (Y=pyrimidine, W = A or T, N= any nucleotide. N is +1 initiation site) - TATA box (-20 to -30 upstream) and CCAT box (-75 upstream). can also be GC box, upstream or downstream promoter
2 structural features of the DNA/RNA sequences at the end of the gene that use this type of termination mechanism (rho-independent)
- near the 3' end of the transcript sequence there are two regions that are GC rich and complementary to each other, which pair with each other and form a stem-loop structure which disrupts elongation process - 3' to the stem loop region there is also a run of A residues (8 U residues on the RNA) which facilitates detachment from DNA template due to weak base-pairing
How does RNA synthesis occur via Pol II in eukaryotes
- template strand is in a groove of the polymerase while other strand is outside. The first 8 nucleotides added are associated with the DNA, then the enzymes feed the transcript out along the CTD for processing - template DNA exits and re-associates with other strand of DNA
The 5'-AAUAA-3' sequence in RNA is a signal for two things:
1) endonuclease to remove nucleotides 11-30 bases downstream of this signal and 2) the poly(A)tail polymerase to add up to 300 adenosyl nucleotides to the 3' end.
bacterial and eukaryotic transcription of which of the following DNA sequences would be slowest? 5' CCGGCAATAT 3' 5' AATGCCGAAT 3' 5' TAAGCCACTA 3' 5' GGCGAAATAC 3'
5' CCGGCAATAT 3', because bacteria and eukaryotic transcription is slowest through regions rich in G and C because they form more hydrogen bonds, and require more energy to unwind
RNA polymerase is best described as a
DNA dependent RNA polymerase
What are promoters
DNA sequences located in the 5' region adjacent to the transcriptional start site.
summary; how does elongation via RNA polymerase occur
DNA strands separate, template is then available to base pair with nucleotides. Sigma factor detaches polymerase and moves down DNA. DNA unwinds for transcription and rewinds afterwards
What is required for the polymerization reaction catalyzed by RNA polymerase
DNA template, NTPs, Magnesium
Polycistronic operons can be found in which of the following? I. Heart muscle cell II. Escherichia coli III. Saccharomyces cerevisiae IV. Clostridium perfringes
II and IV Polycistronic operons are transcribed in prokaryotic organisms, as these operons contain many genes that are transcribed together.
If the template strand has a cytosine residue what will be added to the RNA molecule during transcription
GTP
A TATA box can be found in the promoter region of which of the following? I. Heart muscle cell II. Escherichia coli III. Saccharomyces cerevisiae IV. T4 bacteriophage
I and III TATA boxes are common structural features in eukaryotic organisms, which include heart muscle and Saccharomyces cerevisiae. Escherichia coli is a bacteria and T4 phage is a bacterial virus.
Acetylation of histones occurs on which amino acid residue type?
Lysine (K) is a common amino acid in histone protein and the positive charge on this amino acid favorably interacts with the negatively charged phosphodiester bond in DNA, thus stabilizing the structure. Acetylation of this amino acid disrupts the structure and opens part of DNA for transcription
What are ribonucleoside 5' triphosphate used for
RNA polymerase begins copying from a specific point on one strand of the DNA using a special type of sugar containing nucleotide called ribonucleoside 5' triphosphate to begin the growing chain. Additional ribonucleoside monophosphate are incorporated into the growing RNA chain
Which of the following statements concerning bacterial transcription is true? ANSWER RNA transcription and protein translation occur roughly simultaneously in bacteria. Bacterial mRNA have unusually long lifetimes. Bacterial mRNAs are degraded by the spliceosome. Bacterial mRNAs are monocistronic.
RNA transcription and protein translation occur roughly simultaneously in bacteria, since these events occur in the same cellular location
Similarities between transcription and DNA replication
RNA, like DNA, is synthesized in the 5' to 3' direction, so it is antiparallel to the DNA template. Ribonucleotides are added to a growing strand by the RNA polymerase. The specific rNTP that is added is a complementary base to the template strand
TBP
TBP: TATA binding protein binds to TATA "box", bends DNA
transesterification reaction (gene splicing reaction)
The 2' -OH of the A residue attacks the 5' phosphate of the G nucleotide at the splice site. Phosphodiester group is transferred from 3' end of exon sequence to A nucleotide of intron, this is called the branch point
monocistronic
The coding pattern of eukaryotes in which one mRNA molecule codes for only one protein.
Transcription factor names?
Transcription factors are named for the RNA polymerase in which they association; transcription factor 1 associates with RNA polymerase I, TFII with RNA polymerase II and so on
Which of the following subunits in the E. coli RNA polymerase is responsible for specificity? σ α β β' ω
The σ subunit of RNA polymerase in E. coli recognizes specific primer sequences and recruits the RNA polymerase. Without this subunit, RNA polymerase binds promoter regions randomly
Eukaryotic RNA polymerase II requires which proteins for selective binding?
Transcription factors
What sequences are recognized by the RNA polymerase
Two conserved sequences at the 10 and -35 region are recognized by the sigma factor of the RNA polymerase, though the RNA polymerase covers a region from -55 to -5 in the promoter area
What is a transcription bubble
When RNA polymerase moves, it will unwind the DNA to open up the template and rewind it after transcription of that particular region is complete. Approx 18 bp of DNA are unwound within the polymerase, called a transcription bubble. The bubble occurs occurs during transcription when a small portion of DNA double strand is unwound. RNA polymerase binds to the exposed DNA and synthesizes a new RNA strand. mRNA bound to template DNA and fed out back of the RNA polymerase complex.
Rho protein
a helicase that unwinds DNA-RNA duplexes. In this case, rho protein binds to a specific rho recognition sequences on the RNA transcript, a C-rich region where the RNA polymerase also seems to pause because of high G-C base pair content. Rho unwinds the RNA-DNA duplex and releases the transcript
how to form a lariat structure
attack 5' end by branch point. -OH to form lariat structure. Cleavage of 3' occurs, release of "lariat" RNA intron gets degraded
What begins transcription initiation events
binding of RNA polymerase holoenzyme at promotor sites
What does the RNA polymerase holoenzyme bind to
binds loosely to the -35 promoter and binds tightly to the -10 promoter and untwists. After the promoter region is unwound, there are two strands accessible to be used as a template for transcription
TFIIB
binds to TBP and DNA and recruits polymerase and opens DNA
branch site in mRNA splicing
branch site is 7 nucleotides long with an A group
rNTP
building blocks of RNA synthesis as well as the synthesis of primers in DNA replication.
RNA polymerase CTD
carboxyl terminal domain (CTD) contains repeated 7-mer, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain regulates transcription and recruits protein that cap, splice and Poly A the RNA transcript. In eukaryotes
What is alternative splicing?
different combination of exons from the same gene are combined together. Different proteins can be encoded for only one gene; allows for tissue specific differences. The mature mRNAs would then possess different coding sequences and undergo translation into different proteins. Enlarges the number of possible proteins that can be produced when human genome only has ~20,000 genes
Acetylation of histones __________ DNA binding, and therefore __________ the rate of transcription.
disrupts; increases Lysine (K) is a common amino acid in histone proteins and the positive charge on this amino acid favorably interacts with the negatively charged phosphodiester bond in DNA, thus stabilizing the structure. Acetylation of this amino acid disrupts the structure and opens part of DNA for transcription. This destabilization increases the rate of transcription.
What is mRNA
during transcription, bases are added onto the mRNA strand that are complementary to the template strand. The template is the strand that is complementary to the "coding" strand. The resultant RNA will look like the coding strand, except the T's will be replaced with U's (and they will be ribonucleotides, not deoxyribonucleotides)
In eukaryotes, the 5'-AAUAA-3' sequence in RNA is a signal for __________ to __________.
endonuclease; remove nucleotides from the 3' end
Energy for RNA polymerase reaction from?
energy for the reaction is provided by hydrolysis of the incoming triphosphate molecule (incoming nucleotide) Pyrophosphate is released when a new nucleotide triphosphate is added onto 3' hydroxyl group of the previous nucleotide, which forms a phosphodiester bond. The pyrophosphate is subsequently hydrolyzed in an exergonic reaction providing energy for the process
Eukaryotic vs. prokaryotic polymerases
eukaryotes have three polymerases instead of one in prokaryotes, each which transcribe a specific class of RNAs. For each polymerase, several proteins must assemble at the promoter and other upstream sites, along with the polymerase to form a transcriptional complex.
Genetic organization of eukaryotic genomes
eukaryotic mRNA is monocistronic. Each mRNA only encodes the information for one protein. Eukaryotic genes also have distinctive post-transcriptional modifications, such as a methylated 5' guanine cap and a polyadenylated tail on the 3' end of the message. internal sequences modified by splicing
Why is eukaryotic transcription more complex in terms of genes that are transcribed and those that are not when compared to prokaryotic transcription
eukaryotic transcription is much more complex in terms of the genes that are on and off. need to deal with chromatin structure. use transcription factors instead of a sigma factor to recognize promoter sequences. TFI, TFII, TFIII.
splice sites mRNA splicing
exons and introns are marked by specific sequences at splice sites
describe formation of the transcription initiation complex
factors bound at sequences far from promoters can influence transcription by inhibiting or activating it
The "transcription bubble" is formed during which stage of transcription?
formed during the elongation phase where the core enzymes unwind a short segment of DNA, separate the two strands, and catalyze polymerization off of the template ssDNA strand.
What determines where transcription will begin and which gene will be transcribed?
initiation sequences in the DNA
What are the distinct phases of transcription
initiation, elongation, and termination
What determines where RNA polymerase holoenzyme sits on a promoter
interaction between specific proteins and specific DNA base sequences
intron splice sites
introns begin with GU and end with AG (consensus sequence is longer than this)
In the post-transcriptional processing of eukaryotic pre-mRNA, __________ are excised by the __________.
introns; spliceosome
Disctinctive post transcriptional modifications of eukaryotic genes
methylated 5' guanine cap, polyadenylated tail on the 3' end of the message
What do transcription factors do in eukaryotes
necessary to recruit and bind polymerase to the correct location.
RNA polymerase reaction
new nucleotides are added onto the 3' - OH group. Reaction is highly exergonic, cleaving off the PPi. Major difference from DNA, doesn't need a primer
exons vs. introns
primary RNA transcript is turned into mRNA. Exons encode amino acids while introns do not. RNA splicing removes introns, linking together protein coding regions
Polycistronic message
prokaryotic genomes are organized into blocks of functionally related genes, can be polycistronic. One RNA transcript is translated into many protein. Tend to be related to common functions (Operons)
RNA pol 1 synthesizes
rRNA
CTD regulated by what
regulated by phosphorylation. If it is not phosphorylated, elongation won't occur (TFIIH)
spliceosomes
small nuclear ribonucleoprotein particles (snRNPs) containing small nuclear RNAs (snRNAs)
During splicing, __________ of the spliceosome will base pair with a(n) __________ in pre-mRNA.
snRNA; intron-exon boundary
spliceosome action in splicing
spliceosome action is useful to mediate cleavage and transfer to branch point. mutations in splice sites can cause incorrect mRNA formation and truncated proteins
Which of the following is/are function(s) of the poly(A) tail processing of eukaryotic mRNA?
stabilizing the mRNA and facilitating mRNA entry into the cytosol
RNA Pol III synthesizes
tRNA
Considering the zinc finger motif (shown below), which is a common motif in DNA binding proteins like RNA polymerase III, which part of the structure will bind directly to DNA? The alpha helix The beta-sheet The zinc ion The double helix
the alpha helix binds perfectly into the major grooves of DNA
What gives promoter specificity to prokaryotic RNA polymerase (RNAP)
the dissociable sigma subunit. no specific promoter binding if sigma subunit isnt bind; with the sigma unit bound there is specific promoter binding and weak non-specific DNA binding
What does eukaryotic transcription machinery need to account for specifically?
transcription machinery has to account for a more complicated chromatin structure, specifically DNA complexed with histone proteins.
How does termination of transcription occur in eukaryotes
transcription proceeds past the end of the gene in eukaryotes. In processing pre-mRNA, cleavage of transcript occurs just past the AAUAAA signal. Poly A tail added, which is important for mRNA stabilization and transport from nucleus to cytoplasm