Transcription

transcription

synthesis of RNA molecules from a DNA template

RNA polymerases

Large enzymes that catalyze transcription

What are the substrates for RNA polymerase

ATP, GTP, CTP, UTP, aka ribonucleotide triphosphates.

What are the requirements for RNA polymerase function

DNA template strand, promoter sequence, Mg2+ ions

What are the three stages of RNA synthesis

Initiation, elongation, and termination

RNA polymerase catalyzes the formation of ________

phosphodiester bond. nucleophillic attack of 3 OH on the last nucleotide on the alpha phosphoryl group od the incoming NTP, releasing a pyrophosphate

What do the mg2+ ions do for RNA polymerase

one remains tightly bound, one comes in with the NTP and leaves with the pyrophosphate

Transcription bubble

Complex of double stranded DNA that has been locally unwound in a region of 17 base pairs

in what direction is RNA polymerized

5’ to 3’

Template strand (antisense strand)

has a sequence that is the complement of the RNA transcript, the one that is read by RNA polymerase

Coding strand (sense strand)

Has the exact same sequence as the RNA transcript except for T in place of U

core enzyme

Bacterial core RNA polymerase (alpha2betabeta’w)

holoenzyme

forms when the sigma subunit joins the core enzyme

what is the starting site of trancription, bubble open spot, termed as

+1

what are the two promoters in bacterial transcription

-10 sequence and -35 sequence upstream from the start site

How does only one RNA polymerase function in prokaryotes

multiple sigma subunits see different promotors

Promoter

DNA sequence where RNA polymerase binds to begin transcription. Marks transcription start site and usually upstream of the gene.

Strong promoter

promoters on genes that are transcribed frequently, have high affinity for RNA polymerase, aligns closely to the ideal binding sequence for RNA polymerase

weak promoter

promoters for genes that are transcribed less frequently, do not bind RNA tightly, have -10 and -35 sequences that have substitutions making them less appealing to RNA polymerase

Upstream element

Located 40 to 60 nucleotides upstream the transcription site, increases effeciency of transcription by adding an additional interaction site for polymerase

How does a transcription bubble formed in bacterial transcription

RNA polymerase combines with sigma unit to form holoenzyme. The holoenzyme binds the promoter, with the sigma factor recognizing promoter DNA. DNA remains double stranded, polymerase is attached but DNA is not unwound yet. RNA polymerase then unwinds DNA near the -10 region where it is rich in A and T base pairs, creating the transctiption bubble. RNA polymerase does not need a helicase, it does the unwinding itself.

Does RNA synthesis need a promoter

no

What are the 3 steps of elongation in bacterial transcription

1. rNTP binds in the RNA polymerase active site and forms a base pair with the template strand

2. the 3’ OH group of the growing RNA chain, oriented and activated by Mg2+, attacks the alpha phosphoryl group, forming a new phosphodiester bond and displacing PPi

3. the RNA-DNA hybrid shifts to bring the 3’ end of the newly added nucleotide into proper position for the nect nucleotide to be added

what is the backtracking function of bacterial RNA polymerase

If an incorrect base pair is placed, polymerase will slide backwards, stopping RNA synthesis, RNA 3’ end is displaced from the active site, a segment of RNA sticks out the enzyme. Hydrolysis is used to break the RNA phosphodiester bond, removing the incorrect RNA nucleotide

What are the two mechanisms by which bacterial transcription is terminated

p-dependent chain termination and intrinsic chain termination

Intrinsic termination

RNA polymerase reaches terrminator sequence in DNA and transcribes a GC-rich palindrone, followed by A rich part in DNA that creates a U rich segment in RNA. Because of the GC rich palindrome, the RNA folds back on itself, base pairs internally, making a hairpin loop. The hairpin loop causes polymerase to pause, and the weak U-A base pairing destabilized the RNA DNA hybride. Paused polymerase and weak A-U bonds cause RNA to detach from RNA template, and RNA polymerase dissociates

P dependent chain termination

p protein detects termination signals and binds to synthesized RNA at C rich site. Uses ATP to translocate downstream on the RNA strand. RNA polymerase pauses at terminator region, giving p time to catch up. Once p reaches the transcription bubble, it breaks bonds between RNA and DNA, releasing RNA and ending transcription.

how is bacterial transcription regulated

E coli has seven distinct sigma factors for recognizing promoter sequences. For example, one recognizes a standard promotor, a heat chock promoter, and nitrogen starved promoters.

What are riboswitches and how do they function

Riboswitches are in the 5 untranslated region of an mRNA, that can bind to a small metabolite ligand and change its shape to stop further elongation of the mRNA

ribozyme

an RNA molecule that can act as an enzyme and catalyze a biochemical reaction

what strand of DNA is transcribed

template

both p dependent and p independent chain terminatino of transcription are alike in that

they both manage to dislodge the RNA transcript from the template

How are tRNAs and rRNAs processed after transcription in prokaryotes

Transcripts can be cleaved at specific sites, nucleotides can be added to the termini of chains, bases and ribose units can be modified

How does eukaryotic transcription differ from bacterial

Transcription is not coupled to translation in eukaryotic, transcription occurs in the nucleus in eukaryotes and cytoplasm in prokaryotes, eukaryotes have 3 polymerases while prokaryotes have one, rna polymerase binds promoter with sigma in prokaryotic but eukaryotic need transcription factors, prokaryotes do not process their mrna while eukaryotes do, chromatin needs opened in eukaryotes and it is free in prokaryotes

polycistronic mRNA

commonly found in prokaryotes, several genes are encoded by the same RNA molecule, each gene has its own start and stop codon.

how is chromatin organized in eukaryotes

DNA is wrapped around histone proteins to compact and organize DNA, some genes are readily accesible while others arent. eukaryotic gene expression needs the manipulation of chromatin structure

What are the three types of RNA polymerases in Eukaryotic transcription

RNA polymerase I, RNA polymerase II, RNA polymerase III

RNA polyermase I

Located in the nucleolus, transcribes most rRNAs

RNA polymeras II

In the nucleoplasm, transcribes mRNA and snRNA

RNA polymerase III

In nucleoplasm, transcribes tRNA and 5s rRNA

What are the elements found in genes transcribed by RNA polymerase II

TATA box, initiator element, downstream core promoter element, CAAT box and GC box

TATA box

30 base pairs upstream of start site, served as binding site for transcription factors that recruit RNA polymerase II. TATA binding protein binds, bending DNA, recruiting transcription factors

Initiator element

found at transcriptional start site and defines the start site

Down stream core promoter element

found downstream of start site at +28 and +32, recruit transcription machinery when there is no TATA box or to further support the TATA box

CAAT box

Conserved promoter sequence -75 from start site, binds transcription factor proteins and enhances promoter strength

GC box

GC rich promoter found -90 to -40 upstream, serves as site for activator proteins and found in a lot of housekeeping genes, enhances transcription activation

TFIID

First transcription factor to bind to the promoter, contains TATA binding protein and TAfs that help recognize Inr, DPE, and CAAT. TFIID recognizes the core promoter DNA sequences and positions RNA polymerase II correctly, and recruits other transcription factors

TFIIH

Has helicase activity, unwinding promoter DNA near start site to create the transcription bubble, it also phosphorylates RNA polymerase II allowing it to move from the promoter and begin elongation

What are enhancer sequences, what are their properties, what are their functions

Cis acting elements that increase the activies of promotors, can stimulate the promoter over distances of thousands of nucleotides, can be upstream, downstream, or in the middle of a gene, fold over and bind to transcription factor complex

How is the the primary transcript modified in eukaryotes

5’ cap, polu A tail, splicing of introns

What is the 5’ cap and how is it added

The 5’ cap is a terminus at the 5’ end containing 5’-5’ triphosphate linkage. It protects the 5’ end from phosphatases and nucleases, and enhanhes mRNA translation. The 5’ end is immediatley modified, with the release of a phosphoryl group. The diphophate 5’ end attacks 5’ end of GTP, the guanine then gets methylated

What is the poly A tail and how is it added

The poly A tail is a string of 250 As at the 3’ end that is added by poly(A) polymerase after cleavage of the pre-mRNA by an endonuclease

What is the general mechanism of RNA splicing by the spliceosome complex

spliceosome is a large complex of sRNAs and proteins (snRNPs). Each intron has 3 important sites: 5’ splice site, branch point, and 3’ splice site. U1 binds 5’ splice site, U2 binds to A in branch point. rest of snRNPs assemble to form the spliceosome. The branch point adenine attacks the 5’ splice site through transesterfication, cutting the RNA at the start of the intron. The intron loops into a lariat shape. U5 aligns the free 5’ exon with the 3’ exon, and the second transesterfication occurs. Results in mature splices RNA

Retinitis pigmentosa

disease of aquired blindness, due to a mutation in pre-mRNA

What is the significance of alternative splicing

generates protein diversity and changes in the coding sequence

how does alternative splicing occur

trans acting splicing factors bind to premRNA , different combinations of exons from the same gene may be spliced into mature RNA

What is an example of catalytic RNA

Group 1 self splicing introns. These are introns that can excise themself and need guanosine as a cofactor. Splice sites are aligned with the catalytic residues by base pairing between the IGS in the intron and the 5’ and 3’ exons.

genes that are inactive are ______ compacted

highly

what does transcription of eukaryotic DNA require

RNA polymerase II and binding of several proteins to stabilize

In eukaryotes ___ enables the same primary transcrip to produce two different proteins

alternative splicing

what transcribes mRNA in eukaryotes

RNA polymerase II

polycistronic mRNA comes from

prokaryotes

why isnt RNA synthesis monitered as highly as DNA

mistakes in RNA are temporary and less harmdul, mRNA only lasts for a short time, so it disapperas quickly. would only make a few faulty proteins, but so many other mrna strands would mask the impact of one or two faulty proteins made from the rna. rna is not heritable either unlike dna

In alternative splicing, there are ____ possible outcomes

2

what direction does RNA polymerase read

3’ to 5’

snRNA

complex involed in the splicing of a primary transcript

rRNA

combines with proteins to form ribosomes

tRNA

brings amino acids to mRNA during protein synthesis

mRNA

carries genetic information to the ribosome for protein synthesis

miRNA

helps regulate the expression of specific genes

sigma factor

a protein that binds to bacterial promoters and forms the holoenzyme

enhancer

DNA sequence with no promoter activity on its own but can greatly increase the activity of promoters