Transcription (Lecture 2)

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transcription

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-performed by RNA polymerase which is large

-primer is NOT required

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RNA structure

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classes of RNA

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1. ribosomal RNA (rRNA)

2. messenger RNA (mRNA)

3. transfer RNA (tRNA)

4. small nuclear RNA (snRNA)

5. micro RNA (miRNA) + small interfering RNA (siRNA)

6. ribozymes

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rRNA

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-make up ribosomes along with ribosomal protein subunits; site of protein assembly

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mRNA

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-carry coding instructions from DNA --> ribosomes; specifies sequence of amino acids; derived from pre-mRNA after processing

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tRNA

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-brings amino acid to match the coding triplet in the mRNA to elongate the polypeptide chain; reads language of amino acids and nucleic acids

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snRNA

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-combine with small nuclear protein subunits to form small ribonucleoproteins (snRNPs)

-process pre-mRNA --> mRNA

-exon splicing

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miRNA + siRNA

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-eukaryotic cells

-degrade or inhibit further translation of mRNA

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transcription unit

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-region of DNA that codes for an RNA molecule

1. promoter: determines which strand to transcribe, transcription apparatus recognizes/binds, determines transcription start site (first nucleotide to transcribe into RNA)

2. RNA-coding region: DNA sequence copied into RNA

3. terminator: DNA sequence that signals transcription to end; termination occurs after the terminator sequence has been copied

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DNA template

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-transcription takes place on only one DNA strand (replication takes place on both)

-template strand: DNA transcribed

-non-template strand: not transcribed

-a gene can only be transcribed from one strand, but all genes don't reside on one strand; both will be used

-promoter dictates which one

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RNA synthesis

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-RNA molecule is formed that is complementary to the template DNA strand

-no primer

-5' --> 3'

-complementary and antiparallel

-U instead of T

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prokaryotic RNA polymerase

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-ALL transcription sets are carried out by RNA pol

-large enzyme consisting of several polypeptide chains

-core RNA polymerase molecule consists of 5 subunits that catalyze elongation: alpha, alpha, beta, beta', omega

-other subunits join and leave the core during transcription

1. sigma factors: control the binding of RNA pol to the promoter

2. rho factors: facilitate transcription termination

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other subunits of RNA polymerase

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-join and leave the core during transcription

1. sigma factors: control the binding of RNA pol to the promoter

2. rho factors: facilitate transcription termination

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prokaryotic initiation

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-promoter recognition

-sigma subunit of RNA polymerase: binds to promoters

-prokaryotic promoters:

1. TATAAT (Pribnow box, 10 bases upstream of the start site)

2. TTGACA (35 bases upstream)

-RNA polymerase combines with sigma subunit (holoenzyme) at the +1 site (transcription start site)

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holoenzyme

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-core protein (RNA poly) + sigma subunit = fully functioning enzyme

-combine at +1 (transcription start site)

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prokaryotic elongation

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-sigma subunit dissociates and RNA polymerase continues elongation

-moves downstream

-RNA polymerase unwinds the DNA (no helicase like in replication)

-topoisomerase: relieves supercoiling ahead and behind the transcription bubble

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prokaryotic termination

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-occurs due to the transcription unit recognizing a termination sequence

-RNA is released from RNA poly

-dissociation of RNA and DNA template

-two types:

1. rho-dependent termination

2. intrinsic termination

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rho-dependent termination

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-able to cause termination only in the presence of rho factor protein

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intrinsic termination

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-able to terminate without rho factor

-nothing external involved, only DNA sequences that are transcribed are involved

-two components

1. inverted repeats: when transcribed into RNA, forms a hairpin loop (complementary base-pair to each other)

2. six adenine nucleotides

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eukaryotic vs prokaryotic transcription

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1. multiple RNA polymerases in eukaryotes

2. nucleosome structure: packaging of DNA

3. promoter binding and initiation differs

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types of eukaryotic RNA polymerases

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-RNA poly I: rRNA (ribosomal)

-RNA poly II: pre-mRNA, snoRNAs, snRNAs (small nuclear and small nucleolar)

-rRNA poly III: tRNA

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nucleosome structure

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-due to eukaryotic chromatin structure, chromatin remodeling is needed to access the DNA

-acetyltransferase protein adds acetyl groups to amino acid ends of histone proteins

-destabilizes the nucleosome structure

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eukaryotic promoter binding

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-promoter recognition: carried out by accessory proteins that bind to promoter

1. general transcription factors: forms basal transcription apparatus with RNA polymerase (low/base level of transcription)

2. transcriptional activator proteins: bind to specific recognition sequences, communication with environment (responding to exntl stimuli like pathogen, heat, stress), enhancing/boosting transcription level, beyond basal rate

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RNA poly II promoters

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-close to transcription start site

-core promoter: immediately upstream of coding sequence (TATA box -30bp, TFIIB recognition element -35bp)

-regulatory promoter: immediately upstream of core promoter (CAAT box, GC box, OCT box)

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enhancers************************

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-further away from transcription start site (>2kb)

-sequences that increase rate of transcription

-transcriptional activators bind to enhancer

-can be upstream or downstream of the coding sequence or inside an intron

-can involve DNA loops to bridge physical distance between different transcription factors and RNA poly II

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eukaryotic initiation************************

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eukaryotic elongation

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-same prokaryotic

-several nucleotides are linked

-RNA polymerase leaves the promoter and transcription factors

-moves downstream

-5' --> 3'

-RNA base pair with DNA template

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eukaryotic termination

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-RNA poly I (rRNA): uses termination factor like rho in bacteria and is sequence specific

-RNA poly III (tRNA): transcribes a terminator sequence similar to intrinsic termination in bacteria

-RNA poly II (mRNA): not sequence-specific, pre-mRNA is transcribed past the coding sequence needed; mRNA is cleaved at a specific coding sequence to generate 2 segments of pre-mRNA

-trailing segment: enzyme Rat1 attaches to 5' end

-exonuclease activity degrades mRNA until it reaches RNA polymerase

-beginning segment continues to mRNA-processing