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Operator, operon, regulon, promoter, enhancer, silencer, insulator, ribosome binding sites, initiator, attenuator???!

posted Jun 23, 2011, 1:15 PM by Rajiv S   [ updated Jun 27, 2011, 6:50 AM ]
Which is which and what is what?

Operon:

An operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter.


We talk about the lac operon for example...


A promoter is the set of sequences to which the RNA polymerase binds to initiate transcription. 

What are Promoters ?

promoter is a regulatory region of DNA located upstream (towards the 5' region) of of a gene, providing a control point for regulated gene transcription.

The promoter contains specific DNA sequences that are recognized by proteins known as transcription factors. These factors bind to the promoter sequences, recruiting RNA polymerase, the enzyme that synthesizes the RNA from the coding region of the gene.

PROMOTER ELEMENTS

1. Core promoter - the minimal portion of the promoter required to properly initiate transcription

  • Transcription Start Site (TSS)
  • Approximately -34
  • A binding site for RNA polymerase
  • General transcription factor binding sites

2. Proximal promoter - the proximal sequence upstream of the gene that tends to contain primary regulatory elements

  • Approximately -250
  • Specific transcription factor binding sites

Difference between Eukaryotic and Prokaryotic Promoters

Prokaryotic promoters

In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site.

  • The sequence at -10 is called the Pribnow box, or the -10 element, and usually consists of the six nucleotides TATAAT. The Pribnow box is absolutely essential to start transcription in prokaryotes.
  • The other sequence at -35 (the -35 element) usually consists of the six nucleotides TTGACA. Its presence allows a very high transcription rate.

Eukaryotic promoters

Eukaryotic promoters are extremely diverse and are difficult to characterize. They typically lie upstream of the gene and can have regulatory elements several kilobases away from the transcriptional start site. In eukaryotes, the transcriptional complex can cause the DNA to bend back on itself, which allows for placement of regulatory sequences far from the actual site of transcription. Many eukaryotic promoters, contain a TATA box (sequence TATAAA), which in turn binds a TATA binding protein which assists in the formation of the RNA polymerase transcriptional complex. The TATA box typically lies very close to the transcriptional start site (often within 50 bases).

 



What about ribosomes? What about DNA polymerase? Where do they bind to, to start anything? 

Ribosome binding sites:


Ribosomes bind to ribosome binding sites: the Shine Dalgarno sequence in proks. and the Kozak sequence in euks:

Protein synthesis is regulated by the sequence and structure of the 5' untranslated region (UTR) of the mRNA transcript. In prokaryotes, the ribosome binding site (RBS), which promotes efficient and accurate translation of mRNA, is called the Shine-Dalgarno sequence after the scientists that first described it. This purine-rich sequence of 5' UTR is complementary to the UCCU core sequence of the 3'-end of 16S rRNA (located within the 30S small ribosomal subunit). Various Shine-Dalgarno sequences have been found in prokaryotic mRNAs (see Figure for the consensus sequence). These sequences lie about 10 nucleotides upstream from the AUG start codon. Activity of a RBS can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator AUG.

In eukaryotes, the Kozak sequence A/GCCACCAUGG, which lies within a short 5' untranslated region, directs translation of mRNA. An mRNA lacking the Kozak consensus sequence may be translated efficiently in Ambion's in vitro systems if it possesses a moderately long 5' UTR that lacks stable secondary structure. Our data demonstrate that in contrast to the E. coli ribosome, which preferentially recognizes the Shine-Dalgarno sequence, eukaryotic ribosomes (such as those found in retic lysate) can efficiently use either the Shine-Dalgarno or the Kozak ribosomal binding sites.


Consensus RBS Sequences. The +1 A is the first base of the AUG initiator codon (shaded) responsible for binding of fMet-tRNAfMet. The underline indicates the ribosomal binding site sequence, which is required for efficient translation.


Operator:

In the Operon Model, the operator is the gene segment to which a repressor binds. This prevents the RNA polymerase from transcribing certain genes in the operon unit.




Enhancer DNA sequences bind transcription factors called enhancer-binding proteins which increase the rate of transcription. Enhancer sequences may be kilobases away from the gene they influence. An enhancer complex may interact with promoter complexes by bringing the sites into direct contact. >> by contact of the enhancer with RNApol complex.. transcription is initiated.. a bit like a switch..

Silencer DNA sequences are the opposite to enhancer sequences. They decrease or suppress the rate of transcription.

Insulator DNA sequences are located between enhancer and silencer sequences and, as there name suggests, they prevent a gene from being transcribed non-specifically by another gene's enhancer.>> wow!!!


And what about DNA polymerase where does it bind?

Origin of replication

From Wikipedia, the free encyclopedia

The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated.[1] This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses. DNA replication may proceed from this point bidirectionally or unidirectionally.

The specific structure of the origin of replication varies somewhat from species to species, but all share some common characteristics such as high AT content. The origin of replication binds the pre-replication complex, a protein complex that recognizes, unwinds, and begins to copy DNA.


Pre-replication complex

From Wikipedia, the free encyclopedia

pre-replication complex (pre-RC) is a protein complex that forms at the origin of replication during the initiation step of DNA replication. The proteins involved in the pre-RC are essential for DNA replication.


http://dnareplication.csc.mrc.ac.uk/research.html


Introduction
The DNA Replication Group studies the assembly and function of the DNA replication machine. Accurate duplication of the genome is a prerequisite for life. In humans, mistakes originating from DNA replication can cause cancer and many other diseases. The focus of the Group is to understand the DNA replication machine using biochemical, biophysical and structural methods. We are particularly interested in the processes governing the initiation of DNA replication, which are central to DNA replication, silencing of chromatin and growth control. Using this approach we intend to discover essential reactions in DNA replication as future anti-cancer drug targets.

DNA replication origins
Saccharomyces cerevisiae has the best characterised replication origins. These origins were first identified by their ability to support the replication of mini-chromosomes or plasmids, giving rise to the name autonomously replicating sequences (ARS) elements. Each budding yeast origin consists of a short (~11 bp) essential DNA sequence, called the ARS consensus sequence (ACS), that represents part of the origin recognition complex (ORC) binding site. Besides the ACS, origins also contain B1 and B2 elements. B1 is part of the ORC-Cdc6 recognition sequence and B2 is involved in loading mini-chromosome maintenance (MCM) proteins to DNA. The ACS is essential and the B elements are important. However, if all B elements are mutated they become essential as well. In other eukaryotes, including humans, the DNA sequences at the replication origins vary and no consensus sequence has been identified; instead, any DNA sequence that has a certain length can replicate.

Pre-replication complex (pre-RC) formation in Saccharomyces cerevisiae
 Click to enlarge
Pre-RC formation [+]


Click to enlarge
Pre-IC assembly [+]
The process of pre-replication complex (pre-RC) formation is cell cycle-regulated and occurs only during late M-early G1 phase when cyclin-dependent kinase activity is downregulated by the anaphase promoting complex (APC). The schematic diagram on the right illustrates the main steps in pre-RC formation. Click on the diagram to enlarge it. 1) ORC is bound to replication origins throughout the cell cycle. This interaction is ATP dependent. 2) During G1 phase of the cell cycle, Cdc6 binds to ORC-DNA in an ATP dependent reaction. ORC and Cdc6 will only form a complex in the presence of origin DNA. 3) The MCM complex is the major DNA helicase in eukaryotic organisms. ORC and Cdc6 represent an interaction surface for Cdt1 – MCM. ORC-Cdc6 binding promotes opening of the MCM ring, so it can encircle DNA. 4) Cdc6 ATP hydrolysis promotes closing of the MCM ring and release of Cdt1 and Cdc6. 5) Orc1 ATP hydrolysis promotes release of ORC from the MCM2-7 complex. Now MCM2-7 can slide on DNA. It is unclear if MCM2-7 is loaded on ss- or ds-DNA.

Pre-initiation complex (pre-IC) assembly
Pre-RC formation requires the absence of CDK activity in G1. Once the pre-RC is formed, S-CDK and DDK activity trigger origin firing. At the same time CDK activity destroys origin competence and in consequence new pre-RCs cannot form anymore on DNA. This kinase-dependent switch guarantees that each origin fires only once and is required for genomic stability. At the same time, kinase activity promotes formation of a pre-initiation complex (pre-IC) and eventually DNA synthesis. The assembly of the pre-IC is illustrated in the schematic diagram on the right. Click on the diagram to enlarge it. The essential targets of S-CDK are Sld2 and Sld3. Phosphorylation of Sld2 and Sld3 promotes their binding to Dbp11, which is required for the loading of the DNA polymerase onto origin DNA. MCM’s are likely the essential target of DDK, since a Mcm5 mutant, bob1, bypasses the requirement for DDK activity. DDK phosphorylates specifically loaded MCM2-7 and results in a structural change of unknown function. Moreover DDK activity is required for Cdc45 and GINS binding to the pre-IC. A consequence of CDK and DDK activity is that RPA binds to the origin, which implies that ss-DNA is generated during pre-IC formation. It is believed that the DNA helicase is involved in generation of ss-DNA, since an MCM ATPase mutant fails to efficiently attract RPA to chromatin. Currently, the exact functions of Cdc45, Sld3, GINS and most of the other pre-IC proteins are not known.

Future research
In the future we want to understand the function of the ORC-Cdc6 in loading MCM proteins onto DNA and the role of DNA itself in the loading reaction. Once MCMs are loaded onto DNA we want to uncover their functions on DNA and the mechanisms that regulate their activity. We are interested in determining the 3D structure of the complexes, since this will help to elucidate the function and mechanism of the proteins. Our long-term goal is to understand how the cell duplicates DNA and how this process is misregulated in cancer.








Chromosome Replication

We identified the four components of the GINS complex in our systematic degronscreen of budding yeast essential proteins of previously unknown function. Recently, we have shown that GINS is essential for the activation of “pre-Replication Complexes” (or pre-RCs) that are built at every origin of DNA replication during the G1-phase of the cell cycle (Masato Kanemaki – seeKanemaki and Labib (2006) ).

Chromosome Replication

The pre-RC contains an inactive form of the MCM helicase; activation occurs subsequently during S-phase when two kinases, Cdc7 and CDK, promote the recruitment of GINS to origins. GINS then allows MCM to associate stably with another protein, Cdc45, which is thought to be an essential component of the active helicase.

Loading of Cdc45 and GINS is a complex process that also requires another factor Sld3. We have shown that Sld3 is displaced from the origin during the loading reaction, indicating that it only acts during initiation, and is not required for the elongation stage of chromosome replication. In contrast, GINS and Cdc45 travel with MCM as part of the nascent replisome ( Kanemaki and Labib (2006) ).

During 2005 we identified “Replisome Progression Complexes” (or RPCs) that probably control the advance of eukaryotic DNA replication forks (Agnieszka Gambus and Alberto Sanchez-Diaz – see Gambus et al (2006) ). RPCs are built around the MCM helicase at origins during the initiation of chromosome replication. GINS is a key component of RPCs as it allows other important factors to associate with MCM; these include factors that control the progression of forks past protein-DNA “barriers” (Arturo Calzada and Ben Hodgson – see Calzada et al (2005) ), allow checkpoint activation, and that couple the progression of forks to other processes such as the establishment of cohesion between sister-chromatids. Studies of RPCs are still at a very early stage and there are many interesting and important questions to be addressed. We want to understand the structure, assembly, and disassembly of RPCs, which only exist at DNA replication forks. Both Cdc7 kinase and CDK are required to build the RPC, and the relevant phosphorylations and their consequences remain to be characterised. We would like to understand in molecular detail how RPCs control the advance of DNA replication forks, and how RPCs themselves are regulated to ensure that the progression and stability of forks are preserved from initiation to termination.  







Repressor and activator:

A repressor physically blocks RNA pol
from initiating transcription.


An activator interacts with RNA pol
to promote transcription.

Regulon:

A group of genes that is regulated by the same regulatory molecule. The genes of a regulon share a common regulatory element binding site or promoter. The genes comprising a regulon may be located non-contiguously in the genome.



Attenuator:


http://en.wikipedia.org/wiki/Attenuator_%28genetics%29

The attenuator plays an important regulatory role in prokaryotic cells because of the absence of the nucleus in prokaryotic organisms. The attenuator refers to a specific regulatory sequence that, when transcribed into RNA, forms hairpin structures to stop transcription when certain conditions are not met.




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