Lecture 3 - Exam 2 Flashcards
What is transcription?
Transcription is the first step in gene expression. It is the synthesis of RNA and DNA.
Transcription and translation are?
Coupled and highly regulated.
Why is it important for transcription and translation to be couple?
To protect the mRNA from breaking down, but also these bacteria grow so quickly that if it is being transcribed, then it also needs to be translated right there and then.
The expression of the genetic material occurs in two consecutive steps:
Transcription: DNA -> mRNA
Translation: mRNA -> protein
Multimeric proteins consist of several ______.
Each of these might be encoded by a distinct region of ______.
Subunits ; DNA
What is a “Cistron” or gene?
A region of the DNA that codes for a single polypeptide.
What is monocistronic mRNA?
Codes for a single protein.
All eukaryotic mRNA is monocistronic.
What is polycistronic mRNA?
Encodes two or more proteins.
Polycistronic mRNAs are common in prokaryotes.
A genetic operon contains….?
Multiple genes transcribed as a polycistronic mRNA.
The gene products of an operon have closely related functions and are similarly regulated.
UTR =
untranslated region
Describe RNA.
Uracil instead of thymine.
2 degree structures: stem-loop and pseudoknots
RNA is prone to modifications and processing.
What are the three main classes of RNA in prokaryotes?
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)
What are the properties of RNA polymerase?
No primer needed (e.g. with lagging strand DNA replication), helicase activity not needed, slower rate than DNA polymerase (1000 nt/s for DNA polymerase vs. 50 nt/s for RNA polymerase).
What are some of the other RNA molecules?
Small RNAs (sRNA), CRISPR RNAs (crRNA), and Riboswitches
Describe small RNAs (sRNA).
Are produced in bacteria and can regulate gene expression.
This can happen in different ways, for example:
Inhibiting translation by binding to complementary mRNA close to the ribosomal binding site.
and
Regulating mRNA stability by influencing RNAse degradation.
Describe CRISPR RNAs (crRNA).
Are complementary to a bacteriophage genome sequence and guide the Cas9 endonuclease to digest viral nucleic acids, providing a defense against viral attack.
Describe Riboswitches.
Are gene control elements that directly bind to specific ligands to regulate gene expression without the need for proteins.
Riboswitches change gene expression by mRNA destabilization or by affecting ribosome binding. This provides negative feedback of gene expression when a metabolite is present.
What do Riboswitches consist of?
- An aptamer domain, usually in the 5’ UTR that senses a metabolite (i.e., lysine riboswitch)
- An expression platform, which changes its conformation when the metabolite binds.
Transcription: Carried out by ________.
RNA strand in made in the __________ direction.
RNA polymerase ; 5’ to 3’
Transcription: The strand of DNA that RNA polymerase uses as a template is called the “_______,” the _________ or the _______.
Sense strand ; the coding strand ; the minus (-) strand
Transcription: The non-template strand is called the _________, or the _______.
Antisense strand, or plus (+) strand.
Transcription: In bacteria, all RNA are synthesized by…?
A single RNA polymerase composed of a core enzyme that associates with a sigma factor (Gives the polymerase its specificity).
Transcription: The RNA polymerase enzyme and the RNA product can essentially…?
The RNA polymerase enzyme and the RNA product can essentially rotate around the helix, so there is no need for the helicase that is essential for DNA replication.
RNA polymerase can start synthesis with/without a primer?
WITHOUT A PRIMER!!! NO PRIMER NEEDED.
Transcription is more complex or simpler than replication?
SIMPLER!!
What are the three transcriptional phases?
- Initiation
- Elongation
- Termination
Describe the first transcriptional phase, initiation.
RNA polymerase recognizes the promoter region upstream from the gene to be transcribed.
RNA polymerase binds at the promoter region and begins to unwind the DNA strands.
Describe the second transcriptional phase, elongation.
RNA polymerase moves along DNA template strand adding nucleotides to the 3’ end of the RNA.
Describe the third transcriptional phase, termination.
RNA polymerase reaches the termination site sequence and the newly-transcribed RNA is released.
What are the multiple polypeptide subunits that comprises the single enzyme RNA polymerase?
The core enzyme and the sigma factor.
What is the core enzyme comprised of?
Two identical alpha chains and two unique beta chains (B and B’)
Describe the sigma factor of a RNA polymerase.
Determines the specificity of promoter binding. Sigma factor confers transcriptional specificity so that RNA polymerase begins to synthesize RNA from an appropriate initiation site (initiating transcription and required for promoter sequence recognition).
What is a holoenzyme?
The core enzyme and sigma factor combine to yield the functional holoenzyme.
All subunits of the core enzyme are _________ in __________ among bacteria, and homologs of the core subunits are also present in Eukarya and Archaea.
highly conserved ; sequence and structure
Describe the size of the RNA polymerase and what it covers.
The RNA polymerase is a very large enzyme and covers the entire promoter (the region upstream of the gene to be transcribed).
The promoter of the gene (or operon) to be transcribed has two regions, what are they?
-10 and -35 nucleotides upstream of the start site, called the consensus sequence.
What is the major sigma factor?
Sigma 70.
Major sigma factor can recognize the consensus sequence because these sequences are very similar in all promoters.
Why are the -10 and -35 regions crucial?
The -10 and -35 regions are crucial because mutations that affect promoter function usually occur in one of these regions.
For any bacterium, there is a rough correlation between the number of its….?
Genes that encode sigma factors and the diversity of environments that it experiences.
Ex: If you have a super specialized bacteria, it will have less sigma factors because it will be in one place with a few things.
What is a promoter? Where do they exist?
A DNA sequence onto which the transcription machinery (RNA pol) binds and initiates transcription.
Generally, promoters exist upstream of the genes they regulate.
Why is the specific sequence of a promoter very important?
Because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently.
What leads to the localized unwinding of the DNA strands which allows the RNA polymerase to move down the template strand, making an RNA copy, beginning at the start site (or the +1 position!!!)
Recognition of the consensus sequence by the sigma factor and binding of the RNA polymerase to these sequences is what leads to all that.
Within the promoter lie…? What are they particularly important for?
Within the promoter lie two 6 base pair sequences that are particularly important for promoter function and are highly conserved between species.
At the -10 and -35 regions upstream of the initiation site, there are …?
Two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species.
The -10 sequence has the consensus…? Which is important for? What is another name for it?
The -10 sequence has the consensus TATAAT.
This is an important recognition site that interacts with the sigma factor of RNA pol.
Also called Pribnow box.
The -35 sequences has the consensus…? Which is important for?
TTGACA.
It is important in DNA unwinding during transcription.
Transcription factors can bind upstream (UP) elements and _____________.
Affect transcription initiation.
Is the sigma factor required for the whole process of transcription?
No.
RNA polymerase holoenzyme binds to the ______ region, the initial closed complex is converted to an ________ _________ with two separated DNA strands.
Promoter ; open complex
RNA polymerase holoenzyme binds to the promoter region, the initial closed complex is converted to an open complex with two separated DNA strands. What does this expose?
This exposes the bases of the coding strand, allowing base-pairing of the ribonucleoside triphosphates for synthesis of the RNA.
RNA polymerase holoenzyme binds to the promoter region, the initial closed complex is converted to an open complex with two separated DNA strands. This exposes the bases of the coding strand, allowing base-pairing of the ribonucleoside triphosphates for synthesis of the RNA. After the first few phosphodiester bonds are formed, what happens?
The sigma factor dissociates from the polymerase complex.
The sigma factor is only required for…?
The core enzyme alone is required for…?
Sigma factor is only required for promoter recognition and transcription initiation.
The core enzyme alone is required for extension of the RNA strand (elongation).
Why is it important that the sigma factor does not stay with the polymerase complex the whole time?
It would slow down how much transcription will occur because it is only needed to initiate transcription.
A short region of the newly formed RNA remains base-paired to the DNA template, which does what?
It keeps the DNA strands from re-associating, and permits continued RNA synthesis, until a termination signal is reached.
Once the termination sequence is recognized, what happens?
The newly-synthesized mRNA and the RNA polymerase are released.
Recent research has shown that many bacteria and archaea have the same promoter sequences that can be recognized by _________, and genes can be transcribed from the ____ and _____ DNA strands ________.
two RNA pols ; top & bottom ; simultaneously
Sigma 70 (RpoD) is the primary sigma factor for most of the genes expressed during _______ growth.
Exponential.
Alternate sigma factors exist that regulate..?
The transcription of environment or growth-specific genes, such as when iron or nitrogen is not available, or during heat stress or starvation.
E. coli has how many sigma factors? How are these sigma factors described?
7 and each sigma factor is described based on its molecular weight.
Again, sigma 70 is the primary sigma factor, or “________” sigma factor, in E. coli and has a molecular weight of..?
Housekeeping.
70 kDa
Sigma 70 is used for?
Sigma 70 is used for transcription initiation of all unconditionally essential genes and most genes whose product is required in all growth conditions.
What are the two classes of sigma factors?
- The sigma 70 class are all similar to sigma 70 type sigma factors and are involved in transcriptional regulation of many kinds of responses to environmental signals or cellular stress. Essential and recognize thousands of promoters.
- A distinct class know as the sigma 54 class evolved separately, requires ATP and are involved in nitrogen utilization.
The sigma factor has how many domains? What are they? Where do each of these bind?
There are 4 domains: Sigma 1, sigma 2, sigma 3, and sigma 4.
Each domain binds to a specific sequence of a promoter.
-10 and -35 have a consensus sequence for _____?
Sigma subgroup 2 binds to?
Sigma subgroup 4 binds to?
Sigma 70 ;
-10 region ;
-35 region
The upstream (UP) element does what?
Increases promoter strength.
The UP element is bound by?
The alpha C-terminal domains of the alpha subunits.
Promoters with extended -10 sequence will have sigma ___ and ___ bind -10 and extended regions.
2 & 3
Describe the archaeal RNA polymerase.
The Archaeal RNA polymerase is similar to eukaryotic RNA polymerase II.
~12-13 subunits and no sigma factor for promoter recognition.
Requires a multitude of transcriptional factors to position properly on the promoter.
Regulation of transcription = similar to bacterial mechanism with similar activators and repressors.
What are the factors that affect transcription initiation?
- Promoter strength: Similarity of the promoter to the consensus sequence [-10 (Pribnow box) and-35 (recognition site)]
- DNA supercoiling -> DNA gyrase removes positive supercoiling as in DNA replication.
- Upstream (UP) elements: Binding sites for transcriptional regulators, which can negatively or positively affect transcription initiation.
What are transcription factors?
Transcription factors are proteins that affect transcription, usually by affecting the binding of the RNA polymerase to the promoter.
Transcription Factors: Describe enhancer regions
There may be enhancer regions upstream of the promoter that a transcription factor protein can bind to.
Binding to enhancer regions recruit what? This forms what?
The binding to enhancer regions recruit the binding of the RNA polymerase to the promoter, and forming an open complex.
What are anti-sigma factors?
Are proteins that bind to specific sigma factors and prevent their association with the core enzyme.
The release of the anti-sigma factor can allow rapid activation of those regulated genes, without needing new protein synthesis.
E. coli has about _____ different transcription factors, which can affect…?
350 ;
Can affect transcription even when the levels of sigma factors are not changed.
There are many transcriptional repressors. What are transcriptional repressors? What is an example?
Proteins that bind a promoter region and blocks RNA polymerase from proceeding.
Such as: LexA regulating the SOS response by binding to the SOS box upstream of genes involved in DNA repair.
Again, what are the steps of initiation in transcription?
- Formation of closed complex: Binding of RNA polymerase to the promoter region.
- Formation of open complex: Unwinding of DNA at the -10 (~30 bases long)
- Transcription start: +1
- Initiation ends after formation of the first RNA:DNA bond
When, approximately, does the sigma subunit disassociate from the RNA pol complex?
After addition of approximately 12 nucleotides to the growing RNA strand, the sigma subunit has disassociated from the RNA pol complex and the polymerase is moving forward from the promoter region, elongating RNA.
Transcription: Describe the elongation complex.
In the DNA duplex, there is an opening (~18 bp) called the transcription bubble.
In the transcription bubble the elongating RNA forms an RNA/DNA hybrid (8-9 bp). This RNA/DNA hybrid helps keep the RNA Pol attached to the DNA.
The average rate of elongation 40-50 nucleotides per second (vs 1,000 nt/sec for replication).
The rate of elongation of mRNA matches the rate of _______.
What does this require?
Protein synthesis (approx. 16 amino acids per second). This requires mRNA to move through the ribosome at a rate of 48 nucleotides (16 codons) per second.
Ribosomal RNA is synthesized at ______ nucleotides/second in rapidly growing cells.
90.
Describe Factor-Independent Transcription Termination.
(Talk about what a terminator is & what happens when an RNA polymerase encounters a terminator)
Terminator - short sequence that is complementary to the sequence preceding it, frequently followed by a run of Us.
When RNA polymerase encounters a terminator it will transcribe it into RNA, with the consequent formation of a stem-loop.
A stem loop causes the transcription bubble t close, which will hinder the activity of the RNA polymerase.
RNA tends to dissociate from the DNA template, thereby terminating mRNA synthesis. In other cases, termination is sometimes dependent on the activity of the Rho Factor (or other factor dependent terminators).
Describe Factor-Dependent Transcription Termination.
(Talk about the factor that causes termination, where that factor will bind to, and how termination finishes)
The Rho protein binds to the rut sequences (a Rho utilization site) when the RNA polymerase reaches a ‘pause’ site.
A hexamer of Rho proteins encircles the single-stranded RNA and moves rapidly in the 5’ to 3’ direction.
The Rho protein then physically pulls apart the RNA:DNA hybrid.
This dissociates the RNA polymerase core enzyme from its DNA template and terminates transcription.
In bacteria, replication and transcription occur _________ and use the same template.
Simultaneously
Gene transcription in bacteria is biased in the genome. What does this mean?
Most genes are transcribed and replicated in the same direction (to avoid head-on collisions).
Polymerases use the same template at different rates, or moving in opposite directions. What can happen?
This can cause problems. Generally, co-directional collisions do not result in serious DNA damage.
Head-on collisions can cause replication fork breakdown and significant DNA damage.
