Assemble the DNA Follow base pair rules Blue—Guanine Red—Cytosine Purple—Thymine Green--Adenine.
Chapter 13 - Transcription. RNA structure Nucleotides –Ribose sugar – OH at 2′ C Unstable;...
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Transcript of Chapter 13 - Transcription. RNA structure Nucleotides –Ribose sugar – OH at 2′ C Unstable;...
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