Biochem 3-5 Transcription

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transcription

Back 1

RNA synthesis

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messenger RNA (mRNA)

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-Fnx: encode polypeptide sequence
-relative abundance: lowest
-RNA Pol II

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ribosomal RNA (rRNA)

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-Fnx: components of robosomes
-relative abundance: highest
-RNA Pol I

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transfer RNA (tRNA)

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-Fnx: aa carriers
-relative abundance: moderate
-RNA Pol III

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small nuclear RNA (snRNA)

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-Fnx: splicing
-relative abundance: moderate
-RNA Pol III

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small nuclear (5S-rRNA)

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-Fnx: ribosomes
-relative abundance: moderate
-RNA Pol III

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Upstream and downstream is relative to the _____ where transcription starts

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base

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Upstream is __ to the start of transcription, downstream is __.

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5', 3'; during transcription the new RNA strand is synthesized in the 5' - 3' direction, so downstream is in the direction of transcription. An upstream region is in front of the gene.

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transcription numbering system

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The base where transcription starts is numbered +1. Upstream bases are negative, downstream positive (e.g. the base twenty three bp upstream from the start is position -23).

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promoter

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A region of DNA used to activate or repress transcription of a gene. The position of a promoter cannot be moved relative to the gene. The promoters of Pol I & Pol II (rRNA & mRNA) are usually adjacent to the gene on the upstream side. Pol III promoters (e.g. tRNA) are within the gene.

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enhancer

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A region of DNA that regulates transcription like a promoter, but can be moved relative to the gene it controls. The distinguishing characteristic of an enhancer is that its 5'-3' orientation can be flipped in a transgenic expression assay. A promoter cannot. Also the distance between an enhancer and its gene can be altered. Enhancers are often located great distances from their genes, possibly 40 kb or more. Less often they are within a gene, or even downstream.

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minimal promoter

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Smallest region of a full promoter that will drive detectable transcription. Can be thought of as priming transcription. They drive constitutive, low level transcription. They are not responsible for regulation. Upstream sequences of promoters or enhancers are required to elevate, or repress, transcription under specific conditions.

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TATA box

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Most minimal promoters for Pol II possess the consensus sequence TATA[A/T]A

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TATAless promoters

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There are some promoters that lack any sequences similar to the TATA box. These genes tend to be expressed at low levels.

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AT rich sequences

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Adenosines and thymidines only have two specific bonds, while guanosines and cytidines have three. Therefore, DNA strands with AT rich sequences, like the TATA box, are held more weakly. This property may facilitate stand separation during initiation of transcription.

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The Initiation Complex

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- a cluster of proteins known as transcription factors, that assembles around a promoter to initiate transcription

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General Transcription Factors

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the proteins that form the core initiation complex at the minimal promoter. They are called general to distinguish them from the upstream transcription factors responsible for gene specific regulation. These upstream transcription factors bind to the general transcription factors to increase the size of the initiation complex

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TBP (TATA Binding Protein)

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This is the general transcription factor that actually binds the TATA box. Other general transcription factors assemble around TBP to form the core initiation complex at the minimal promoter.

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The Start of Transcription

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This is usually a purine (A > G), 26 to 34 bp downstream from the TATA box. In mammals the consensus around the G/A start is GTTGCTCCT[G/A]AC.

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TBP first binds several general transcription factors in the nucleoplasm. These complexes then find a ____ box to bind.

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TATA; Other general transcription factors are then recruited to form a core initiation complex.

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What must RNA polymerase find 30 BPs downstream from the core initiation complex to start transcription.

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RNA polymerase is atttracted to the core initiation complex and looks for a purine where it will initiate transcription.

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What is the consensus in mammals that RNA Pol must find to initiate transcription?

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a purine (A or G) ~ 30 BP downstream from the TATA box: GTTGCTCCT[G/A]AC... note, the start point is [G/A]

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Upstream Promoters and Enhancers

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Minimal promoters only drive low level, constitutive transcription. Upstream promoter and enhancer sequences are required to elevate and regulate transcription.

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Recognition sequences

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Promoters and enhancers are actually clusters of recognition sequences that are bound by transcription factors. Recognition sequences are usually short, 4 to 10 bp long. They are often palindromic, which is to say they are composed of inverted, end to end repeats. Note, recognition sequences are consensuses, so they can vary slightly from gene to gene.

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What are the 3most common recognition sequences?

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-TATA box: recog seq for TBP; most common
-CCAAT box: raises baseline transcription; second most common
-SP1: GC region 20-50 bp long; third most common

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What recognition sequence does CP1 transcritpion factor bind? What about SP1 transcription factor?

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CCAAT box, SP1

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What recognition sequence is common in TATAless promoters?

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SP1

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transcription factors

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Proteins that bind recognition sequences to control transcription.

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DNA binding domain

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a region of a transcription factor that recognizes and binds a specific sequence of DNA. DNA binding domains are usually basic, which is to say they are positively charged. This charge forms ionic bonds with the negative charged phosphates on the surface of DNA.

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Activation domain

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the region of a transcription factor that induces RNA synthesis by attracting an RNA polymerase. Activation domains can be on either side of the DNA binding domain. Their position varies even within the same family of transcription factors.

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The Acid Blob Model- Many activation domains are masses of negative charge with little structure and few specific interactions. This negative charge serves to attract polymerases non-specifically. This model is based on four observations:

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1. DNA binding domains have similar sequences in a particular family of TFs; activation domains are variable, random sequences of negatively charged AAs
2. DNA binding domains have specific structures; activation domains are random coils
3. DNA binding domains target specific sequences of of specific genes; activation domains are non-specific
4. RNA polymerases are naturally attracted to the negative charge of DNA. Thus, activation domains serve to concentrate negative charge at a specific site on the chromosome. This targets RNA polymerases to that location.

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Promoters and enhancers are actually clusters of _____.

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recognition sequences

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How do transcription factors "stick" to each other?

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As they bind recognition sequences they become grouped together on DNA; they stick to each other through non-specific ionic and hydrogen bonds.

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Upstream transcription factors contribute to the initiation complex by binding to the general transcription factors at the _____.

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minimal promoter; this makes the initiation complex a big 'ole mess of negative charge, which attracts RNA Pol even more.

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promoter and enhancer architecture

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Transcription Factor Families

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There are four major families of transcription factors. Most are classified based on the DNA binding domains. Remember binding domains within a family have similar sequences, but activation domains are usually variable.

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What are the 4 major transcription factor families?

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-helix-turn-helix
-zinc fingers
-helix-loop-helix
-leucine zippers

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Helix-turn-Helix

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(e.g. Hox Genes, PIT-1)
DNA Binding Domain - two α helices are positioned at right angles to each other, connected by a short linker region. One α helix has a basic face. This binds the DNA by wedging into the major groove.

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Zinc Fingers

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(e.g. TBP , SP1, Steroid Receptors)
DNA Binding Domain - Cysteine and histidine residues are chelated to a central zinc ion, creating several loops in the polypeptide. These loops act like “fingers,” wedging themselves into the major groove to bind DNA.

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Helix-loop-Helix

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(e.g. MyoD)
DNA Binding Domain - Like the HTH factors, helix-loop-helix (HLH) factors have two α helices separated by a linker region. However, the linker regions of HLH proteins are longer, and there is no sequence similarity between HLH and HTH factors. HLH factors form heterodimers, which means that two different polypeptides must bind to each other before they will bind DNA.

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Leucine Zippers

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(e.g. Fos, Jun, Myc, C/EBP, CREB)
Interaction Domain - The leucine zipper protein has of an α helix with a leucine every 7 residues. There are 3.6 amino acids per turn of an α helix, so there is a leucine every two turns. Thus the leucines are aligned on the same side of the helix. Leucine is a hydrophobic amino acid, so this alignment creates a helix with a hydrophobic face. The hydrophobic face of one leucine zipper binds with the hydrophobic leucines of another, and the two factors are held together as a dimer.
DNA Binding Domain – These are extensions of α helices which grip the recognition sequence on each side of the DNA (“scissors grip” binding).

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Primary transcript

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RNAs that have not been processed.
Note, processing commences before transcription is completed, so the two occur simultaneously.

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capping

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a guanosine is added to the first nucleotide of the primary transcript by an unusual 5' - 5' bond.

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5' Cap

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The cap is methylated, as are the 2' hydroxyls of the first and third nucleotides. These three sites are methylated with all mRNAs. Numerous other nucleotides will be methylated along the transcript, although the location of these additional sites vary. Capping and methylation is believed increase mRNA stability by preventing its degradation.

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Splicing

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removal of a section of the primary transcript. The region that’s spliced out is called an intron; regions that are retained are exons. Introns can be over 10,000 bp long. The diagram shows the consensus sequences at each end of an intron.

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Introns and Exons

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Introns are spliced out by spliceosomes. These are complexes of snRNPs (small nuclear ribonucleoprotein particles) (pronounced snirps), which are composed of proteins and snRNAs (small nuclear RNAs). Interestingly, the enzymatic activity resides in the RNA, not the protein. Note, snRNAs are transcribed by Pol III, the same polymerase that produces tRNAs.

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Polyadenylation

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final event of mRNA processing is to add 20 - 300 adenosines to the 3’ end of the transcript. These are known as poly A tails. They are believed to increase mRNA stability. Polyadenylation is signaled by the sequence AAUAAA, but transcription does not stop at this site. It continues for another 500 - 2000 bp before Pol II falls off the DNA. The excess RNA is cleaved at a CA, 10-30 bp downstream of the AAUAAA sequence. After removal the RNA fragment is degraded. Poly A polymerase then produces a poly A tail by adding adenosines to the 3’ end of the transcript. The vast majority of mRNAs are polyadenylated. However, a few are not. Histones are one example of mRNAs that lack poly A tails.

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polyadenylation scheme

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