TRANSCRIPTION AND TRANSLATION & GENETIC CODE

SUMMARY Transcription Translation Mutations Initiation Elongation and termination Translation Mutations

(information storage) (information carrier) (active cell machinery) Transcription (information carrier) Translation (active cell machinery)

Transcription is the process of creating an equivalent RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA in the presence of the correct enzymes. During transcription, a DNA sequence is read by RNA polymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. Transcription is the first step leading to gene expression. The stretch of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene.

Transcription can be explained easily in a quick 5 step explanation. Step 1: DNA unwinds/"unzips" as the Hydrogen Bonds Break. Step 2: The free nucleotides of the RNA, pair with complementary DNA bases. Step 3: RNA sugar-phosphate backbone forms. (Aided by RNA Polymerase.) Step 4: Hydrogen bonds of the untwisted RNA+DNA "ladder" break, then the RNA leaves the nucleus through the small nucleur pores. Then goes to the cytoplasm to continue on to protein processing.

Transcription process Pre-initiation In eukaryotes, RNA polymerase, and therefore the initiation of transcription, requires the presence of a core promoter sequence in the DNA. The most common type of core promoter in eukaryotes is a short DNA sequence known as a TATA box, found -30 base pairs from the start site of transcription. The TATA box, as a core promoter, is the binding site for a transcription factor known as TATA binding protein (TBP), which is itself a subunit of another transcription factor, called Transcription Factor II D (TFIID). Five more transcription factors and RNA polymerase combine around the TATA box in a series of stages to form a preinitiation complex. 1.Core Promoter Sequence 2.Transcription Factors 3.DNA Helicase 4.RNA Polymerase 5.Activators and Repressors. The transcription preinitiation in archaea is essentially homologous to that of eukaryotes, but is much less complex.

Initiation In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA. RNA polymerase is a core enzyme consisting of five subunits: 2 α subunits, 1 β subunit, 1 β' subunit, and 1 ω subunit. At the start of initiation, the core enzyme is associated with a sigma factor (number 70) that aids in finding the appropriate -35 and -10 base pairs downstream of promoter sequences. Transcription initiation is more complex in eukaryotes. Eukaryotic RNA polymerase does not directly recognize the core promoter sequences. Instead, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription. Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it. The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription initiation complex.

Elongation One strand of the DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy. Although RNA polymerase traverses the template strand from 3' → 5', the coding (non-template) strand and newly-formed RNA can also be used as reference points, so transcription can be described as occurring 5' → 3'. This produces an RNA molecule from 5' → 3', an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).

Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly produced from a single copy of a gene. Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind.

Termination Bacteria use two different strategies for transcription termination. In Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C rich hairpin loop. When the hairpin forms, the mechanical stress breaks the weak rU-dA bonds, now filling the DNA-RNA hybrid. This pulls the poly-U transcript out of the active site of the RNA polymerase, effectively terminating transcription. In the "Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex. Transcription termination in eukaryotes is less understood but involves cleavage of the new transcript followed by template-independent addition of As at its new 3' end, in a process called polyadenylation.

Non-template (coding) strand DNA 5 3 5 RNA 3 3 5 Template strand Phosphodiester bond is formed by RNA polymerase after base pairing occurs 3 5 Template strand 3 5 RNA 5 3 Hydrogen bonds form between complementary base pairs DNA template 3 5

Transcription Sequence in prokaryotes Initiation phase RNA polymerase binds a Sigma protein that in turn binds to the promoter, upstream from the start site for a particular gene on DNA. Sigma opens up a portion of the DNA helix and RNA polymerase begins transcription.

1. Initiation begins Promoter (on non-template strand) 35 box 10 box HOW TRANSCRIPTION BEGINS Promoter (on non-template strand) 35 box 10 box Upstream DNA +1 site Sigma Active site Downstream DNA RNA polymerase 1. Initiation begins Sigma binds to promoter region of DNA.

2. Initiation continues Template strand Non-template strand +1 site HOW TRANSCRIPTION BEGINS Template strand Non-template strand +1 site RNA NTPs 2. Initiation continues Sigma opens the DNA helix; transcription begins.

Transcription Sequence in prokaryotes cont’d Elongation and termination phase Elongation: RNA polymerase adds nucleotides 5’ to 3’ at a rate of ~ 50 nucleotides/second beginning at start site. Active portion = transcription bubble. Completed mRNA strand exits bubble as it is finished. Termination: RNA polymerase stops when the RNA produces a hairpin loop.

HOW TRANSCRIPTION ENDS Upstream DNA Hairpin loop RNA polymerase RNA RNA Downstream DNA DNA Transcription termination signal 1. RNA polymerase reaches a transcription termination signal, which codes for RNA that forms a hairpin. 2. The RNA hairpin causes the RNA strand to separate from the RNA polymerase, terminating transcription.

C A G U OH 3’ 5’

Transcription Sequence in eukaryotes is basically the same; 3 differences: 3 types of RNA polymerase – Promoters are more complex and include sites for basal transcription factors + others.

Transcription Sequence in eukaryotes – differences cont’d Posttranscriptional modifications Addition of a 5’ cap (adenine or guanine + methyl-GTP and a “poly A tail”.

5 cap 5 3 Poly(A) tail 5 untranslated region Coding region

Translation Translation is the third stage of protein biosynthesis (part of the overall process of gene expression). In translation, messenger RNA (mRNA) produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein. Translation occurs in the cell's cytoplasm, where the large and small subunits of the ribosome are located, and bind to the mRNA. The ribosome facilitates decoding by inducing the binding of tRNAs with complementary anticodon sequences to that of the mRNA. The tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome in a fashion reminiscent to that of a stock ticker and ticker tape.

Initiation phase Translation in Prokaryotes A small sequence of rRNA on the ribosome binds to a complementary sequence on the mRNA with the help of intiation factors. *The start codon, AUG is exposed. tRNA with a sequence that is complementary to the codon (=anticodon) attaches to the codon and releases its amino acid.

Early model of aminoacyl tRNA function Amino acid 3 Binding site for amino acid 5 Binding site for mRNA codon Serine anticodon 5 3 mRNA Serine codon

The Code A specific order of nucleotides or bases on the DNA. Occurs in blocks of 3 bases = codons that specify which amino acid goes where in a protein. 1 codon = one amino acid The code is “universal”, i.e. the codons specify the same amino acids in all organisms, pretty much.

Elongation and termination phase Ribosome moves down 1 codon at a time and specific tRNA’s bring their amino acids to the chain. Amino acids are joined by peptide bonds to form the protein. 3 sites on the ribosome (APE): A = tRNA binding site, P = site of peptide bond formation, E = exit site for empty tRNA’s.

Diagram of ribosome during translation Polypeptide grows in amino to carboxyl direction (amino acids in green) Peptide bond formation occurs here Aminoacyl tRNA Large subunit Anticodon mRNA 5 3 Codon Small subunit The E site holds a tRNA that will exit The P site holds the tRNA with growing polypeptide attached The A site holds an aminoacyl tRNA

Prokaryotes cont’d Elongation and termination phase Translation is terminated when the ribosome reaches a stop codon.

ELONGATION OF POLYPEPTIDES DURING TRANSLATION Ribosome Peptidyl site tRNA Exit site Aminoacyl site mRNA 5 3 5 3 5 3 Start codon 1. Incoming aminoacyl tRNA New tRNA moves into A site, where its anticodon base pairs with the mRNA codon. 2. Peptide bond formation The amino acid attached to the tRNA in the P site is transferred to the tRNA in the A site. 3. Translocation Ribosome moves down mRNA. The tRNA attached to polypeptide chain moves into P site. The A site is empty.

ELONGATION OF POLYPEPTIDES DURING TRANSLATION Exit tunnel Elongation cycle continues 5 3 5 3 5 3 4. Incoming aminoacyl tRNA New tRNA moves into A site, where its anticodon base pairs with the mRNA codon. 5. Peptide bond formation The polypeptide chain attached to the tRNA in the P site is transferred to the aminoacyl tRNA in the A site. 6. Translocation Ribosome moves down mRNA. The tRNA attached to polypeptide chain moves into P site. Empty tRNA from P site moves to E site, where tRNA is ejected. The A site is empty again.

Bacterial ribosomes during translation Multiple ribosomes can translate each mRNA simultaneously Ribosomes DNA In bacteria, transcription and translation are tightly coupled. 5 end of mRNA 3 Ribosome translates mRNA as it is being synthesized by RNA polymerase 2 2 Protein 1 1 1 Ribosome RNA polymerase Start of gene End of gene (3 end of template strand) (5 end of template strand)

mRNA DNA Transcription and RNA processing in nucleus Mature mRNA Mature mRNA Translation in cytoplasm Ribosome Protein

Translation Eukaryotes Eukaryotic genes contain sequences that do not contain codons = INTRONS; sequences that contain codons are EXONS. mRNA sequences contain the same introns and exons.

Micrograph of DNA-RNA hybrid Interpretation of micrograph (c) Genes and RNA transcripts differ in length. Single-stranded DNA only Intron Exon Size of gene (DNA) Single-stranded DNA base paired with mRNA Size of mature RNA transcript

Translation in Eukaryotes Eukaryotes cont’d Introns are removed after mRNA synthesis and exons are joined together = RNA splicing. snRNPs = small nuclear ribonucleoproteins are the splicers. Why? Alternate splicing can produce different proteins from the same gene sequence. 30,000 genes can be used to produce 120,000 mRNA’s.

Introns must be removed from RNA transcripts. DNA 3 5 Promoter Exon 1 Exon 2 Exon 3 Primary RNA transcript 5 3 Spliced transcript 5 3

Genetic code The genetic code is the set of rules by which information encoded in genetic material (DNA or mRNA sequences) is translated into proteins (amino acid sequences) by living cells. The code defines a mapping between tri-nucleotide sequences, called codons, and amino acids. With some exceptions, a triplet codon in a nucleic acid sequence specifies a single amino acid. Because the vast majority of genes are encoded with exactly the same code, this particular code is often referred to as the canonical or standard genetic code, or simply the genetic code, though in fact there are many variant codes.

Translation starts with a chain initiation codon (start codon) Translation starts with a chain initiation codon (start codon). Unlike stop codons, the codon alone is not sufficient to begin the process. Nearby sequences (such as the Shine-Dalgarno sequence in E. coli) and initiation factors are also required to start translation. The most common start codon is AUG which is read as methionine or, in bacteria, as formylmethionine. Alternative start codons (depending on the organism), include "GUG" or "UUG", which normally code for valine or leucine, respectively. However, when used as a start codon, these alternative start codons are translated as methionine or formylmethionine. The three stop codons have been given names: UAG is amber, UGA is opal (sometimes also called umber), and UAA is ochre. Stop codons are also called "termination" or "nonsense" codons and they signal release of the nascent polypeptide from the ribosome due to binding of release factors in the absence of cognate tRNAs with anticodons complementary to these stop signals.

Amino acid

The Genetic Code

Transcription vs. Translation Review Process by which genetic information encoded in DNA is copied onto messenger RNA Occurs in the nucleus DNA mRNA Translation Process by which information encoded in mRNA is used to assemble a protein at a ribosome Occurs on a Ribosome mRNA protein