Module 11 - RNA and Transcription

the flow of genetic information in cells

DNA → transcription → mRNA, rRNA, or tRNA → ribosome (all RNAs) → translation → protein

primary structure of a nucleic acid

the sequence of nucleotides

reminders: RNA has U instead of T and an OH group on the 2- C of its sugar instead of an H (more reactive than DNA)

forms secondary structures when folded up

the flow of genetic information in cells

DNA → transcription → mRNA, rRNA, or tRNA → ribosome (all RNAs) → translation → protein

primary structure of a nucleic acid

the sequence of nucleotides

reminders: RNA has U instead of T and an OH group on the 2- C of its sugar instead of an H (more reactive than DNA)

forms secondary structures when folded up

secondary structure

a structure that results when an RNA primary structure folds up due to hydrogen bonding between complementary bases on the same strand

e.g. a stem-loop (aka hairpin) or an alpha helix

types of RNA in all cells

mRNA

rRNA

tRNA

mRNA

messenger RNA

one of the types of RNA found in all cells

rRNA

ribosomal RNA

ribosomal RNA

one of the types of RNA found in all cells

tRNA

transfer RNA

one of the types of RNA found in all cells

types of RNA found only in eukaryotes

pre-mRNA

snRNA

snoRNA

miRNA

siRNA

piRNA

pre-mRNA

pre-messenger RNA

one of the types of RNA found only in eukaryotes

snRNA

small nuclear RNA

one of the types of RNA found only in eukaryotes

snoRNA

small nucleolar RNA

one of the types of RNA found only in eukaryotes

miRNA

microRNA

one of the types of RNA found only in eukaryotes

siRNA

small interfering RNA

one of the types of RNA found only in eukaryotes

piRNA

piwi-interacting RNA

one of the types of RNA found only in eukaryotes

types of RNA found only in prokaryotes

crRNA

crRNA

CRISPR RNA

the type of RNA found only in prokaryotes

transcription

the transfer of genetic information from DNA by the synthesis of a complementary RNA molecule copied from the DNA template

requires a gene in DNA (the template), RNA polymerase (the enzyme), and free NTPs

primers are not needed

the antisense strand

the template strand of DNA

gets transcribed

bases are complementary to the RNA formed

the bottom dark green strand in the picture

the sense strand

the non-template strand of DNA

does not get transcribed

bases match the RNA formed, except Ts gets swapped for Us

the top dark green strand in the picture

transcription unit

a stretch of DNA that encodes an RNA molecule and the sequences necessary for its transcription

components: promoter, RNA-coding region, terminator

promoter

the binding site for RNA polymerase and the transcription initiation apparatus

one of the components of a transcription unit

depicted as yellow in the picture

RNA-coding region

a sequence of DNA nucleotides that is copied into an RNA molecule

the gene itself; only the transcribed portions of the transcription unit

one of the components of a transcription unit

depicted as blue in the picture

terminator

a sequence of nucleotides that signals where transcription is to end

part of the gene

depicted as red in the picture

phosphodiester bond formation

one phosphate of an NTP (at the 5’ end of a free molecule) attaches to the 3’ OH of another molecule (in a chain)

the H pops off of the OH, two phosphates pop off

during initiation: NTP + NTP → NTP-NMP + P-P

during elongation: NTP-NMPn + NTP → NTP-NMPn+1 + P-P

note: in equation above, - represents a bond between terms, not a minus sign

note: the initial nucleotide of the chain (5’) remains as a nucleoside triphosphate (NTP)

RNA synthesis in transcription

initiation does not require a primer

new nucleotides are added to the 3’ end of the RNA molecule

DNA unwinds at the front of the transcription bubble and then rewinds

consensus sequence

comprises the most commonly found nucleotides at a specific DNA site

a conserved sequence

e.g. TTGACA, TATAAT

e.g Pribnow box in bacteria (TATAAT), which has similar function as TATA box in eukaryotes

found in the promoter

depicted by light yellow in the picture

process of transcription in bacteria

catalyzed by RNA polymerase

sigma factor recognizes promoter, directs RNA polymerase to it → forms holoenzyme

DNA double helix is unwound and denatured locally

initiation of transcription begins at the transcription initiation site

sigma factor dissociates after initation

transcription continues at ~50 nucleotides per second at 37* until the enzyme encounters the terminator sequence

promoter

the binding site for RNA polymerase on the DNA molecule

right before +1

not transcribed

gets recognized by sigma promoter; the site where the sigma factor binds with RNA polymerase

transcription initiation site

where initiation of transcription begins

+1

the first base to be transcribed

sigma factor dissociates here

termination of transcription in bacteria

depends on the formation of the hairpin loop secondary structure at the terminator site

hairpin loop forms after inverted repeats in the gene are transcribed into the RNA molecule and form intramolecular base pairs

formation of the hairpin loop destabilizes the DNA-RNA pairing; transcription ends

can be rho-dependent or rho-independent

rho-dependent termination

rho protein (a helicase) is needed to break the DNA-RNA pairing and terminate transcription

rho binds to an unstructured region of RNA; moves toward its 3’ end

when RNA polymerase encounters a terminator sequence, it pauses, rho catches up

rho unwinds the DNA-RNA hybrid using helicase activity, brings transcription to an end

rho-independent transcription

the weak DNA-RNA pairing at the terminator site is enough to destabilize the interaction and terminate transcription

terminator contains an inverted repeat followed by ~6 A nucleotides

inverted repeats are transcribed into RNA

the string of Us causes RNA polymerase to pause

the inverted repeats in RNA fold into a hairpin loop, which stabilizes the DNA-RNA pairing

the RNA transcript separates from the template, terminating transcription

RNA polymerase II and transcription elongation in eukaryotes

RNA polymerase maintains a transcription bubble during elongation

in the bubble, ~8 nucleotides of RNA remain base-paired with the DNA template strand

the DNA double helix enters a cleft in the polymerase, gets gripped by jaw-like extensions of the polymerase

the two DNA strands get unwound

RNA nucleotides enter the polymerase through a pore, get added to the 3’ end of the growing RNA molecule

the DNA-RNA hybrid funnels through the polymerase, hits a wall and bends → keeps bubble open, positions the DNA-RNA hybrid at the polymerase’s active site

the newly synthesized RNA separates from the DNA, runs through another groove before exiting the polymerase

termination of transcription of protein-coding genes in eukaryotes

does not occur by itself

requires the activity of Rat1 exonuclease

after the protein-coding region of the gene is transcribed, an RNA endonuclease cleaves the RNA molecule at a consensus cleavage site

Rat1 binds to the unprotected 5’ end of the trailing, non-coding RNA fragment, begins to degrade it until it catches up with the RNA polymerase (note: RNA polymerase is still transcribing the DNA)

complete degradation of the trailing fragment terminates transcription

Rat1

a 5’→3’ RNA exonuclease involved in the termination of transcription in eukaryotes

binds to the 5’ end of trailing, non-coding RNA and degrades it until it reaches RNA polymerase

CRISPR RNA (crRNA)

clustered regularly interspaced short palindromic repeats

the basis of a form of adaptive immunity found in bacteria and archaea (prokaryotes)

DNA arrays consisting of a number of short palindromic sequences separated by spacer sequences

3 stages: acquisition, expression, interference

spacer sequences

DNA sequences derived from invading DNA molecules, e.g. bacteriophages or plasmids

separate the palindromic sequences in crRNA

a memory of the invading DNA

three stages of the CRISPR-CAS system

acquisition

expression

interference

acquisition

the first stage of the CRISPR-CAS system

foreign DNA enters the cell; gets identified, processed, and inserted into the CRISPR array as a new spacer

expression

second stage of the CRISPR-CAS system

the entire array gets transcribed into a long CRISPR precursor RNA, then cleaved by a CAS protein into crRNAs

each crRNA contains 1 spacer homologous to a foreign DNA

each crRNA combines with a CAS protein to form an effector complex

interference

the third stage of the CRISPR-CAS system

if the same foreign DNA enters the cell again, effector complex recognizes it by its base-pair complementarity to the crRNA

CAS protein cleaves it with its endonuclease activity