Transcription

What is the central dogma?

The poorly named concept that DNA makes RNA makes protein. The process of making RNA from DNA is translation. The process of making protein from RNA is translation.

What enzyme makes mRNA?

RNA polymerase

What base does mRNA have that DNA does not?

uracil

What is the exception to the central dogma? When does it occur? What enzyme is used?

Reverse transcription. This is done by some viruses, such as retroviruses, which use reverse transcriptase. Some viruses also have RNA genomes and replicate themselves simply by replicating RNA.

When we say mRNA is "copied" do we really mean an exact copy? Can you recognize from an mRNA strand what DNA strand was used to make it?

Not an exact copy, because U substitutes for T. The template strand will be complementary to the RNA strand transcribed from it, and the RNA strand will be identical except for substitution of U for T to the coding strand, the DNA strand that is complementary to the template strand.

Where does transcription occur in eukaryotes? In prokaryotes?

Transcription occurs in the nucleus in eukaryotes. In prokaryotes, it occurs in the cytoplasm since there is no nucleus.

Where are the ribosomes in eukaryotes? In prokaryotes?

In the cytoplasm in both. The implication is that mRNA must leave the nucleus and travel to the ribosome before translation can occur in a eukaryotic cell, while in a prokaryotic cell, translation of a strand of RNA can occur before transcription has even been completed.

Where does transcription start?

Near a promoter

In what direction is mRNA made?

5' to 3'

How many RNA polymerases do we humans have?

3. RNA pol1 works on large ribosomal RNAs. RNA pol2 makes messenger RNAs. RNA pol3 makes transfer RNAs and miscellaneous other small RNAs.

How many RNA polymerases do prokaryotes have?

one

What is the sigma factor?

RNA polymerases are multi-subunit proteins. One subunit is the sigma factor, which helps the other subunits find the promotor. It is only in prokaryotes. The sigma factor is involved in binding to the DNA and helping the RNA polymerase get started in the transcription process.

Why are RNA polymerases more error-prone than DNA polymerases? Why does this not matter?

RNA polymerases lack a proofreading function. They are not under the same evolutionary pressure to avoid mutations because mRNAs are degraded quickly and not passed down through mitosis.

How long does an mRNA typically exist?

minutes to hours

Where do prokaryotes have control sequences located?

10 and 35 nucleotides upstream of transcription start site

What is meant by "upstream" of the start site?

"Upstream" refers to the DNA region located in the 5' direction from the transcription start site. Think of it as being "before" the start site on the DNA strand.

How does the numbering work? What number is the start site?

The transcription start site is designated as +1. Bases upstream of the start site are numbered negatively (-1, -2, -3, etc.), while bases downstream are numbered positively (+2, +3, +4, etc.).

What is meant by consensus sequence?

A consensus sequence is a short DNA sequence that is highly conserved across different genes or species. These sequences often serve as recognition sites for proteins involved in transcription or other DNA-binding processes.

What is a Pribnow box? What factor binds there? What number is it found at upstream?

It's a specific consensus sequence found in prokaryotes, located around 10 base pairs upstream (-10) of the transcription start site. It's essential for the initiation of transcription. The sigma factor of RNA polymerase binds to the Pribnow box.

Do all consensus sequences have the same strength?

No, consensus sequences can vary in their strength, meaning some sequences are bound more tightly by proteins than others. This variation in binding affinity can influence the rate of transcription initiation.

What happens at -35?

Another important consensus sequence in prokaryotes is often found around 35 base pairs upstream (-35) of the start site. This sequence also plays a role in sigma factor binding and transcription initiation.

What do you know about the variety of Sigma factors?

Prokaryotes can have different sigma factors that recognize different promoter sequences. This allows them to regulate gene expression in response to environmental changes by using different sigma factors under different conditions. Heat shock sigma factor is an important example; it turns on a suite of genes that only need to be active when proteins are slightly denatured by excessive heat.

What are the three phases of transcription?

Initiation: RNA polymerase binds to the promoter, unwinds the DNA double helix, and begins synthesizing RNA. Note that the 5' end of the opened DNA hangs off like a tab at the beginning of the 'transcription bubble'.

Elongation: RNA polymerase moves along the DNA template, adding nucleotides to the growing RNA chain.

Termination: RNA polymerase reaches a termination signal, releases the newly synthesized RNA molecule, and detaches from the DNA.

How does transcriptional termination occur? Two ways.

Rho-independent termination: A specific sequence in the DNA template forms a hairpin loop in the RNA transcript, causing RNA polymerase to pause and release the transcript.

Rho-dependent termination: A protein called Rho factor binds to the RNA transcript and travels along it. When Rho factor reaches RNA polymerase, it causes the polymerase to detach from the DNA, terminating transcription. There is no specific point of termination, but areas where termination occurs this way are typically rich in G-C sequences, which slow the polymerase down.

What type of organism has histones?

Eukaryotes.

Why are the histones needed?

Histones are proteins that act as spools around which DNA winds, forming a highly organized and compact structure called chromatin. This packaging is essential for fitting the long DNA molecules within the nucleus.

What are they comprised of?

Histones are positively charged proteins, rich in basic amino acids (lysine and arginine), which allows them to interact with the negatively charged DNA backbone.

What is a nucleosome?

A nucleosome is the basic unit of chromatin, consisting of a segment of DNA wrapped around a core of eight histone proteins.

Which type of DNA is active and which is inactive?

Loosely packed chromatin (euchromatin) is considered transcriptionally active, allowing access to RNA polymerase and other regulatory proteins. In contrast, tightly packed chromatin (heterochromatin) is generally inactive, as the DNA is less accessible.

What are some promoter transcriptional control sequences in eukaryotes?

The TATA box, an A-T rich sequence 10 to 100 nucleotides upstream of the transcription start site.

The CAAT box

The GC box

Which is similar to the Pribnow box?

The TATA box. If a gene has lot of transcription: it will have a TATA box, if very little transcription, probably will not.

How does the transcription initiation complex form?

Before the RNA polymerase can 'sit down' on the DNA in eukaryotic cells, other proteins must first interact with it. Transcription factor 2D (TF2D), a multi-subunit protein, is the first protein that binds to a promoter sequence; one of its subunits, TBP, binds to the TATA box. TF2D facilitates the binding of other proteins, resulting in formation of an open transcription complex. The carboxy terminus of RNA pol2 is phosphorylated

What modifications are performed on new eukaryotic mRNAs?

Before they can exit the nucleus and participate in translation, eukaryotic mRNAs undergo several crucial processing steps:

5' Capping: A modified guanine nucleotide (7-methylguanosine) is added to the 5' end of the mRNA molecule.

3' Polyadenylation: A string of adenine nucleotides (poly-A tail) is added to the 3' end.

Splicing: Non-coding regions called introns are removed, and coding regions called exons are joined together.

What are three reasons why they are "capped" at the 5' end?

Protection from degradation: The cap protects the mRNA from being degraded by exonucleases.

Enhanced translation: The cap helps ribosomes recognize and bind to the mRNA, promoting efficient translation.

Nuclear export: The cap facilitates the transport of the mRNA from the nucleus to the cytoplasm.

What unusual bond is formed?

The 5' cap involves an unusual 5'-5' triphosphate linkage between the 7-methylguanosine and the first nucleotide of the mRNA.

What is the purpose of polyadenylation?

Similar to the 5' cap, the poly-A tail (at the 3' end) contributes to mRNA stability, protects it from nuclease degradation, and enhances translation efficiency.

What are introns and exons?

In splicing, the sequences that are removed are called introns, and the sequences that remain are called exons.

How does splicing work? In what structure does it occur?

Splicing occurs when sequences internal to an RNA are removed and the ends joined together. This forms mature mRNA from the primary transcript RNA. Splicing is carried out by a complex called the spliceosome, which recognizes specific sequences at the intron-exon boundaries and catalyzes the removal of introns and the joining of exons.

What unusual bond is formed here?

Splicing involves the formation of a lariat-shaped intermediate, where the 5' end of the intron is joined to a branch point within the intron. In eukaryotic cells, this almost always involves a GU at the 5' end of the intron joining to an AG at the 3' end. There is a nucleophilic attack that the A makes on the G; an unusual covalent bond between the 5' end of the G and the 2' hydroxyl of the A is formed.

What is alternative splicing and what is its importance?

Alternative splicing is a process where different combinations of exons within a pre-mRNA can be joined together, producing multiple different mRNA isoforms from a single gene. This significantly increases the diversity of proteins that can be produced from a limited number of genes.

Do prokaryotes need all genes transcribed at the same level all the time? Can you think of examples?

No, prokaryotes regulate gene expression in response to their environment. For example, when lactose is present, bacteria might only need to produce enzymes for lactose metabolism.

What do the genes of an operon have in common?

They have the same promoter.

What is a promoter? What happens there?

A promoter is a DNA sequence that serves as the RNA polymerase's binding site, marking the transcription's starting point.

What is a repressor? How does it work? How about an activator?

A repressor attaches to a sequence of DNA called an operator downstream from the promoter, blocking RNA polymerase. An activator binds upstream of the promoter and facilitates/accelerates transcription.

What is an operator? What happens there?

An operator is a DNA sequence located near the promoter that serves as the binding site for repressor proteins.

How does the trp operon work?

The trp operon in bacteria encodes enzymes involved in tryptophan

How would inducer molecules work with other proteins to change gene expression?

binding to repressors or activators.