18 April 2008

So what I am up to?

I'm studying. I haven't pulled an all-nighter in awhile, but cell bio might warrant it. I have such a hard time with it, and now I really want to do well in it. Just in case anyone is interested....here's 1 of 4 of my note sections for the test in 10 hours. Ay yaya...I haven't really been a slacker, but I've been sooo busy. I turned in my thesis to my committee today. Hopefully they won't have too much to change on it! I'm calling it finished :D

Here's a lesson on transcription. Enjoy.... I'll put some new pictures up tomorrow.

Gene Expression

Gene expression is accomplished through proteins. DNA is transcribed into mRNA, which goes to a ribosome (rRNA is a structural component of ribosomes). Then the mRNA is translated to a sequence of amino acids. tRNA is the delivery boy for amino acids to be polymerized into a polypeptide.

The sequence of nucleotides in DNA codes for amino acids which form proteins. AUG is a start codon. Transcription always starts at an AUG.

You all know that DNA is double stranded. For any given gene, only one strand is transcribed into mRNA. This strand is called the template or non-coding strand. Transcription of the template strand generates a complementary code that is similar to the opposite strand of DNA called the nontemplate or coding strand. However, Thymine is replaced by Uracil in RNA.

A set of three nucleotides in mRNA codes is called a codon. There are 4 nucleotides possible meaning there are a total of 64 possible codons (4*4*4). However only 61 code for amino acids. The remaining codons serve as stop codons (UAA, UAG, UGA). The code for amino acids is nearly universal. This code is referred to as degenerate, which means that every codon codes for only one amino acid. Amino acids may be coded for by multiple codons. This offers mutation protection for the organism. The first two bases in a codon are critical; however the remaining codon has more freedom to change w/o changing the AA.

Transcription

Transcription in Prokaryotes is catalyzed by RNA polymerase. Bacterial cells only have a single type of RNA polymerase. It consists of 5 subunits: 2 alpha, 2 beta, and one sigma. The sigma factor is critical for accurate transcription as it promotes RNA polymerase binding to the promoter sequence. The alpha and beta subunits are virtually the same in all RNA polymerase molecules, but the sigma varies depending on the gene category.

The promoter sequence is a specific sequence of DNA that determines where RNA synthesis starts as well as the which strand will be the template strand. mRNA always starts upstream (at a location called the +1 spot) towards the 5’ DNA end. 3’ is called downstream.

The place where transcription begins is called the startpoint/site. Some sequences help identify this spot. Upstream about 10bp (-10bp) there is a sequence known as the Pribnow box (TATAAT). Even farther upstream at -35 there is the -35 sequence (TTGACA). This is an example for one sigma factor. Basically you need to know that sigma factors recognize various sequences specific to that sigma factor.

So… the RNA polymerase binds to the DNA and begins to unwind it in front of it. NTP (ribonucleotide triphosphates) come in and are a part of a polymerization reaction…intiating RNA synthesis. The DNA strand serves as a template and uses NTP as substrates. The promoter determines the direction of transcription. After about 9 nucleotides have been added, the sigma factor (part of RNA polymerase) dissociates. This concludes initiation.

The chunk of DNA that is transcribed is creatively called the transcription unit. After initiation has been started, the RNA continues to elongate through the addition of more NTPs.

RNA polymerase moves along the DNA and unwinds the DNA helix in front of it and adds the complementary nucleotides to the template strand. For a little while a RNA/DNA hybrid exists. RNA is elongated in the 5’ to 3’ direction.

This elongation continues for awhile…until RNA polymerase reaches a termination signal (stop codon). RNA polymerase dissociates and an RNA transcript is born.

When RNA poly runs into a termination sequence, the end of transcription is triggered. There are two types of termination signals in prokaryotes that are classified by their independence or dependence on a protein called a Rho factor. Rho independent termination involves a short chain of GC rich sequence followed by several U residues near the 3’ (tail end) of RNA. Remember the GC bases contain 3 hydrogen bonds (TA has 2) in DNA. So the GC sequence forms a loop that has more bonds (so its stronger) than the 2 Hydrogen bonds between the U of RNA and the A of DNA. When these triple H bonds snap together it essentially rips off the U bonds. Think of a rubber band popping off something. There’s more than one way to skin a cat (please don’t)…and there’s more than one way to terminate RNA synthesis. The second way is Rho dependent. These guys don’t have the GC and U sequence so they need some help. Rho recognizes a specific sequence about 50-90 nucleotides long toward the 3’ (tail) end of RNA. Rho needs energy to act so it requires ATP (ATP-dependent). Once energized, Rho is a helicase (unwinder protein) that unwinds RNA from DNA template –thus releasing it.

Transcription in Eukaryotes (aka…humans, dogs, cats, Betta fish, zebras, pigmy goats, poison arrow frogs, etc).

Of course, Eukaryotes have to outdo prokaryotes so they make things a little more complicated. Eukaryotes have three (3) types of RNA polymerase. Each one uses a different promoter. These promoters can be downstream OR upstream. For RNA polymerase to even bind to DNA, several other factors are required. For binding to occur, protein-protein interactions are VERY IMPORTANT. It is also interesting to note that RNA cleavage is more important than the site of RNA transcription. For example…the gene I’ve done research on has a whole lot of sequence that doesn’t code for protein…but may get put into RNA. Then that sequence is cut out later. So Eukaryote RNA can babble on and on. A later process edits the tape so just what is need is left at a later time. RNA is extensively processed during and after transcription.

Here’s some stuff about the different types. RNA pol I involves ribosomalRNA. II is mRNA. II is tRNA. Mitochondria nd Chloroplasts have their own RNA pol molecules. All these buggers have different promoters – good thing I don’t have to know them!

RNA polymerase I has 2 parts. Remember this is the RNA pol involved with rRNA. The first part is called the core promoter, which is the minimum DNA sequence required for RNA pol I to bind and correctly direct transcription. The CORE is absolutely required for ACCURATE transcription. The other element is called the upstream control element, and, in case you can’t guess, is upstream. The upstream element is required for EFFICIENT transcription. Transcription factors bind to these two locations…then RNA polymerase I binds. He’s like a fat, rich guy (no offense to anyone…) who sends security ahead to secure the place before making an appearance.

And you thought RNA pol I was complicated. Well, RNA polymerase II has 4 types of DNA sequences involved with just the core promoter function. Jumping right in…. first there’s the initiator (Inr) sequence that surrounds the transcription start site. Then there’s the all important ***** TATA box ****** that is located 25 to 30 nucleotides upstream of the start site. The first transcription factor/ protein binds here. This is the RATE LIMITING STEP OF TRANSCRIPTION. Upstream of the TATA box is the TFIIB recognition element (BRE). Then there’s a downstream promoter element (DPE) that is located about 30 nucleotides downstream of the start site (this isn’t in all). The composition of promoters varies…some genes have all, some have none, and some have some combination.

The four of those are categorized into 2 types of core promoters. There are the TATA driven promoters – have an Inr and a TATA (may or may not have the BRE). Then there are the DPE-Driven promoters which have a DPE but lack the TATA box or the BRE.. All by itself, the core promoter supports only basal, minimum transcription. There’s generally upstream elements present that improve efficiency. For example, about 100bp from the startsite of transcription there are some proximal promoter elements… CCAAT box and GCbox (SP1). Summary…5’ to 3’ on DNA = BRE, TATA, Inr (start point), DPE.

RNA polymerase III uses entirely downstream promoters. tRNA and 5s rRNA have two different types of promoters. tRNA promoters contain consenus box A and box B. 5S RNA promoters have consensus box A and box C. These occur actually within the transcription unit..

Transcription factors….These guys are always involved with the transcription of all nuclear genes. A transcription factor is a protein that is required for transcription. YUCK! There are so many of these little buggers. 1st TATA binding protein (TDP = TFIID) binds to the TATA box in the RATE LIMITING STEP…analogous to the sigma factor of prokaryotes. Then TFIIA and TFIIB (BRE binding element) bind. So A,D, and B are bound. Now the big guy RNA Polymerase II can come in and bind (TFIIF is on him).

After RNA pol II has bound…E, and H (helicase) bind too. Now you have A, B, D, E, F, and H bound to DNA along with RNA pol II. F is a protein kinase that phosporylates (energizes RNA pol) then dissociates. Pol II begins moving down the DNA and transcribing. Phew…that’s so complicated and a gross oversimplification, I’m sure.

Termination!! Of course it’s different depending on the RNA pol…

RNA pol I termination occurs when the protein binds to 18 nucleotide termination signal in the RNA chain.

**RNA pol II – cleaved by an endonuclease at a specific site before transcription is even finished/terminated. This occurs 10-35 nucleotides downstream of AAUAAA. OR the cleavage site is where the poly A tail is added to the mRNA.

RNA pol III – short stretch of U residues. There’s no other proteins needed for this sequences to be recognized.

After all this, this is only the primary transcript…which has to go through more modification post transcription. rRNAs are cleaved from a common rRNA precursor. Processing includes removing chunks from the primary transcript and chemical modifications. rRNA is the most abundant/stable form of RNA in a cell. 70-80% of total cellular RNA is rRNA, 10-20% is tRNA, <10% style=""> Ribosomes have multiple typs of rRNA (28S, 18S, 5.8S, and 5S).

28S, 18S, and 5.8S are all encoded by a single transcription unit that is transcribed by RNA pol I and produces a single primary transcript (pre-rRNA). These rRNA are separated by spacer regions. Human haploid genes contain 150-200 copies that are separated by nontranscribed spacer regions on the DNA. After pol I transcription the pre-RNA is cleaved to get rid of the spacers to yield the final, mature rRNAs.

tRNA processing has a lot of stuff that happens too: removal, addition, and chemical modification of nucleotides. There are several dozen types of tRNA…each one brings a particular AA or more to a codon of mRNA during translation. tRNAs are funky little dudes. They all share general structure and are about 70-90nt in length. The have 2 hairpin loops that have complementary base pairing.

tRNAs are processed too. First a 16nt sequence is removed from the 5’ end (head/leader). Two terminal nucleotides are removed from the 3’ (tail) end…and are replaced with CCA. Some ways nucleotides are processed: methlyation, unusual bases (dihdrouracil, ribothymine, pseudouridine, inosine)

mRNA processing generally involves capping (polyA tail) and intron removal. Most of this occurs in the nucleus so the mRNA is ready for translation when it reaches the cytoplasm. Translation (cytoplasm) and transcription (nucleus) in eukaryotes are separated by time and space. The pre-mRNA is often much longer than the final mRNA. Ends are modified. 5’ end has a 5’cap and then they have a poly A 3’ tail. Poly A = lots and lots of Adenine.

The 5’ cap is a methylated at position 7 of the purine ring Guanosine nucleotide. This added shortly after the initiation of transcription. This protects from 5’ nucleases…and helps postion the mRNA on the ribosome. It’s essential for translation initiation.

More..The Poly A tail is normally from 50-250 nucleotides long, but normally is around 200. Present at most 3’ tail ends of eukaryotic mRNA. Histone mRNAs lack a poly A tail. Completed after transcription. Genes do not have long stretches of T. PolyA polymerase catalyzes this addition of polyA sequences to mRNA..independently.

The signal that says add a poly A tail is AAUAAA. The mRNA is cleaved about 10-35 nucleotides downstream of AAUAAA. Poly A protects mRNA from attack at 3’ end, recognized by specific proteins that help mRNA get out of the nucleous, and help the ribosomes recognized mRNA for translation.

Introns are sequences that are within the primary transcript that don’t appear in the RNA. Exons appear in the final functional RNA…Introns are out. Exons stay in.

What removes introns? Splicesomes remove introns from pre-mRNA in a process called RNA splicing. A splicesome is an assembly of an RNA-protein complex known as a snRNP (small nuclear robonucleoproteins…great vocab word, eh?). Remove introns and shove the exons together.

Some introns are self slicing. Suicidal introns are called ribozymes.

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