DNA Transcription

uracil has one less methyl group than

thymine

this makes uracil more unstable, solves the problem of cytosine deamination

since RNA is single stranded

it can fold up into a variety of shape, just like a polypeptide chain folds up to form the final shape of a protein

allows RNA to carry out various functions in cells (structural, regulatory, or catalytic role)

the RNA base pairing includes both conventional and noncoventional base pairing

in both transcription and dna replication

the incoming nucleotide (nucleoside triphosphate) provides the energy needed to drive the reaction forward

rna polymerase

doesn't need primer and doesn't have proofreading mechanism

rRNA

is responsible for the catalytic activity of ribosomes

What is transcription?

Transcription is the process where RNA polymerase synthesizes RNA from a DNA template.

How does RNA polymerase initially interact with DNA?

RNA polymerase collides randomly with a DNA molecule, sticks weakly to the double helix, and slides rapidly along its length.

What happens when RNA polymerase encounters the promoter?

RNA polymerase latches on tightly only after it has encountered the promoter.

Where does bacterial RNA polymerase bind to the promoter?

Bacterial RNA polymerase binds to the promoter specifically at the -10 (aka Pribnow box, called TATAAT) and -35 sequences (TTGACA)

What is the role of the sigma factor in bacterial RNA polymerase?

The sigma factor is a subunit of bacterial RNA polymerase that recognizes the promoter.

What happens to the sigma factor once transcription begins?

Once transcription has begun, the sigma factor is released.

What is the process of elongation in transcription?

During elongation, RNA polymerase moves forward and continues synthesizing RNA.

What signals the end of transcription?

Transcription continues until RNA polymerase encounters a sequence called the terminator.

What occurs after RNA polymerase transcribes the terminator sequence?

The enzyme halts and releases both the DNA template and the newly made RNA transcript.

What does RNA polymerase do after completing transcription?

The polymerase reassociates with a free sigma factor and searches for another promoter to begin the process again.

polarity of promoter orients the polymerase and determines which

DNA strand is transcribed

regions transcribed into RNA contain

the terminator but not the promoter nucleotide sequences

What is the role of the sigma factor in bacteria?

Responsible for recognizing the promoter sequence on DNA.

How does the sigma factor identify the promoter sequence on DNA?

Each base presents unique features to the double helix, allowing the sigma factor to identify the promoter sequence without separating the DNA strands.

What happens when the sigma factor begins to open the DNA double helix?

The sigma factor binds to the exposed base pairs, keeping the double helix open.

initiation of eukaryotic gene transcription

1. general transcription factors and polymerase must assemble at each promoter before transcription can begin

RNA polymerase II

transcribes mRNA

miRNA

snRNA (in spliceosome)

other noncoding RNAs)

transcription initiation complex for RNA polym II described in later flashcards

RNA polymerase I

transcribes rRNA

RNA polymerase III

transcribes tRNA

5S rRNA gene

genes for many other small RNAs

What is the role of general transcription factors in transcription?

Assemble on the promoter.

How do general transcription factors assist RNA polymerase?

They position RNA polymerase.

What do general transcription factors do to the DNA double helix?

They pull apart the DNA double helix to expose the template strand.

What is a similarity between general transcription factors and sigma factors?

Both play a role in initiating transcription.

What is TFIID in eukaryotic transcription?

TFIID is a general transcription factor that binds to the TATA box in eukaryotic promoters.

What is a subunit of TFIID?

The TATA binding protein.

What happens when TFIID binds to the TATA box?

It causes a dramatic local distortion in the DNA double helix.

What is the significance of the distortion caused by TFIID binding?

It serves as a landmark for the subsequent assembly of other proteins at the promoter.

TATA box is located

30 nucleotides upstream the transcription start site

general transcription factors

orient the RNA polymerase so that it will begin transcription in the right direction and on the correct DNA template strand

step 2 of transcription initiation in eukaryotes

binding of TFIID enables adjacent binding of TFIIB

step 3 of transcription initiation in eukaryotes

rest of general transcription factors as well as the RNA polymerase itself then assemble at the promoter

step 4 of transcription initiation in eukaryotes

TFIIH pries apart the double helix at the transcription start point, using the energy of ATP hydrolysis which exposes the template strand of the gene

step 5 of transcription initiation in eukaryotes

TFIIH also phosphorylates RNA polymerase II, releasing the polymerase from most of the general transcription factors so it can begin transcription

step 6 of transcription initiation in eukaryotes

once the polymerase moves away form the promoter, most of the general transcription factors are released from the DNA; the exception is TFIID, which remains bound through multiple rounds of transcription initiation

when transcription is finished

RNA polym II is released

the phosphates on its tail are stripped off by protein phosphatases and polymerase is then ready to find a new promoter (only the dephosphorylated form of RNA polym II can reinitiate RNA synthesis)

at -35

TFIIB binds

at -30 (TATA Box)

TBP subunit of TFIID

transcription start site

TFIID binds

30

TFIID binds

What are the three main steps of RNA processing?

Capping, splicing, polyadenylation (the first and third happens to all pre-mRNAs)

increase stability, allow for protein machine to recognize

When do the steps of RNA processing occur?

As RNA is being synthesized

Where are the enzymes responsible for RNA processing located?

On the tails of RNA polymerase II

phosphorylation of the tail of RNA polym II allows RNA processing proteins to assemble there

RNA capping

adds a guanine and methyl group to the end of pre-mRNA and 5' to 5' triphosphate bridge

polyadenylation

enzyme that cuts RNA chain at a particular sequence

add many adenine nucleotides

exons are shorter than

introns

some genes have no introns, some introns, or many introns

eukaryotic genes

how do spliceosomes know what is an intron?

each intron contains a few short nucleotide sequences that act as cues for its removal from pre-mRNA

found at or near each end of the intron (can be found partially in exons and introns)

snRNPs recognize the special sequences and direct the cleavage of RNA at the intron-exon borders and catalyze the covalent linkage of the exon sequences

splicing machine cuts out the intron in the form of a lariat structure, formed by the reaction of an adenine nucleotide with the beginning of the intron (5' end)

the branch point (A) of the lariat produced in the splicing reaction

branch point closer to 3' than 5'

What are small ribonucleoproteins (snRNPs) composed of?

snRNA + additional proteins

How do snRNPs recognize splice-site sequences?

Through complementary base pairing between their RNA components and sequences in pre-mRNA

What is the function of snRNPs in splicing?

They carry out the chemistry of splicing

What do snRNPs form the core of?

The spliceosome

adenine

How is the intron removed?

A attacks 5' and cuts the sugar-phosphate backbone at this point

the released 5' end of the intron becomes covalently linked to the 2' OH group of the ribose of the adenine to form a branched structure

the free 3' of the exon reacts with start of next exon sequence, joining the 2 exons together

intron is released as lariat structure, later degraded in nucleus

What is the function of U1 snRNP?

Recognizes the 5' splice site

What is the function of U2 snRNP?

Recognizes the lariat branch point site

What is the function of U6 snRNP?

Rechecks the 5' splice site

What triggers the conformational changes in U2 and U6 during splicing?

Hydrolysis of ATP by spliceosome proteins

What does the spliceosome deposit on the mRNA after splicing reactions?

Exon junction complex

What is the purpose of the exon junction complex?

Marks the splice site as successfully completed

RNA synthesis and processing

takes place in factories within the nucleus

these factories contain nucleic acid and proteins (RNA polymerase, accessory proteins that carry out transcription to the enzymes responsible for capping, polyadenylation, and splicing)

proteins required for transcription form loose molecular aggregates, termed intracellular condensates that act as

factories for the production of RNA

What is the function of ribonuclease?

Degrades RNA into nucleotides

Do eukaryotic mRNAs have longer lifespans than bacterial mRNAs?

Yes

What partially controls the lifespan of RNA?

3' UTR

What is a specialized set of RNA-binding proteins that signal a completed mRNA is ready for export?

Cap binding protein, exon junction complex, poly A-binding protein

What guides mRNA through the nuclear pore during export?

Nuclear transport receptor

What happens to mRNA in the cytosol after export?

mRNA sheds some proteins and binds new ones

What role do poly A-binding protein and new proteins play in the cytosol?

They act as initiation factors for protein synthesis

a special signal at the beginning of each mRNA molecule

sets the correct reading frame

What is the typical length of tRNA?

80 nucleotides

What structural feature does tRNA have due to base-pairing?

It looks like a cloverleaf

What type of structure does tRNA have due to additional hydrogen bonds?

L-shaped structure

How many short segments of folded tRNA are double helical?

Four