Chapter 13 - Transcription

RNA structure

• Nucleotides– Ribose sugar – OH at

2′ C• Unstable; short-lived

molecule

– Nitrogenous bases• Adenine• Guanine • Cytosine• Uracil

RNA structure

• Nucleotide polymer held together by phosphodiester bonds

• Usually single-stranded– Due to short regions of

complementary sequences, can base pair to form stems, hairpins, etc

RNA structure

• Primary structure– Nucleotide sequence

• Secondary structure– Formed by

complementary regions

– Has greater variety than helix of DNA

– Various shapes have different functions

Classes of RNA• Ribosomal RNA (rRNA)

– Joins with protein subunits to form ribosomes• Site of polypeptide synthesis

• Messenger RNA (mRNA)– Codes for a polypeptide

• Amino acid sequence– Pre-messenger/primary transcript

• In eukaryotic cells only– Needs to be modified before exiting the nucleus

• Prokaryotic mRNA can start to be translated before transcription is complete

• Transfer RNA (tRNA)– Brings specific amino acid to the ribosome for incorporation into

the growing polypeptide

Classes of RNA cont

• Small nuclear RNA (snRNA)– Joins with small nuclear proteins to form

snRNPs – small nuclear ribonuclear proteins• Assist with post-transcriptional modifications of

primary transcript– Splices out introns

• Small nucleolar RNA (snoRNA)– Aids in the processing of rRNA

Classes of RNA cont

• MicroRNA (miRNA) and small interfering RNA (siRNA)– In eukaryotic cells– RNAi – RNA interference– Initiates degradation or inhibition of mRNA molecules

• Piwi-interacting RNA (piRNA)– Found in mammalian testes– Regulation of sperm development

Synthesizing RNA from DNA

• During DNA replication, the entire DNA molecule is copied

• In transcription, only a small section of DNA is used for the synthesis of RNA– Usually one gene at a time, or several genes (in prokaryotes)

• Only one of the two strands of DNA gets transcribed into RNA– Transcribed/template strand– Nontemplate strand = coding strand

• “coding” strand gives RNA sequence (replace T with U)

Synthesizing RNA from DNA cont• In DNA, one strand may

be the template strand for one gene, while another strand may be the template strand for another gene

• Transcription occurs in the 5′→3′ direction of the RNA molecule– Complementary and

antiparallel to the DNA strand

Transcription Unit• Promotor

– Upstream from coding region– Specific DNA sequence– Serves as attachment site for

transcription molecules– Sequence is NOT transcribed

into RNA

• RNA coding region

• Terminator– Downstream from coding

region– Is transcribed into RNA;

sequence is later removed– Specific sequence to halt

transcription

RNA polymerase• Does NOT require a primer

• Prokaryotic RNA polymerase– Single type of polymerase used for all transcription– Composed of 5 polypeptide subunits – core enzyme– (σ) sigma factor

• Binds with core enzyme to create holoenzyme• Controls binding to promotor

– Without sigma, polymerase will bind anywhere on DNA • Various sigma factors are present for different promotor types• Releases from core protein after transcript is several nucleotides

long

• Eukaryotic RNA polymerase– Different classes for different types of RNA– Consists of multiple subunits

• Core enzyme with accessory proteins at different stages

Bacterial transcription

• Initiation– Specific DNA

sequence at promotor– Consensus sequence

• Most common nucleotides in a particular position

• R = purine• Y = pyrimidine • N = any

Promotor

• Two consensus sequences

• Any change/mutation in promotor region alters the rate of transcription– Down mutation –

reduces rate of transcription

– Up mutation – increases rate of transcription

• rare

Holoenzyme• Binds to promotor consensus

sequences only, but enzyme covers larger area

• Polymerase alters its structure and binds more tightly, unwinding DNA– Begins at -10 sequence and

continues downstream

• Bases on consensus sequence location, enzyme’s active site is in position +1

• First RNA nucleotide is placed complementary to DNA sequence

Elongation

• After transcript is approximately 12 nucleotides long, polymerase structure alters so it is no longer bound to consensus sequences– Moves downstream– Sigma factor is usually released

• Polymerase continues to unwind DNA downstream and rewind upstream– Transcription bubble

• Positive supercoiling ahead of bubble; negative supercoiling behind

– Topoisomerase enzymes relieve tension

Termination – Rho-independent

• Contains inverted complementary sequences that form a hairpin when transcribed– Slows transciption

• 2nd repeat sequence is polyA (polyU on RNA)– Weak (due to 2 H bonds

between each), and transcript separates from DNA template

Termination – Rho-dependent• Rho factor protein

– Binds to regions with no secondary structure

• RNA sequence upstream from termination doesn’t form secondary structure– Rho factor binds to RNA

and moves toward 3′ end

• At a hairpin, transcription slows and rho factor can “catch up” to DNA/RNA– Rho has helicase activity

• Breaks H bonds and separates RNA from DNA

Modifications for eukaryotic transcription

• Nucleosome structure– DNA associated with histone proteins

• Acetylation of histones reduces their positive charge; makes DNA more accessible

• Initiators – Promotors

• Have varied sequences to attract different polymerase types– Polymerases have several associated accessory proteins

• Directly upstream from gene– Enhancers

• Can be located far away from gene• DNA loops around to bring enhancer (with activator protein) to promotor

region• Some sequences can be repressors/silencers

• Termination– Different polymerases have different mechanisms for termination