Transcription and Translation - Lecture Notes | BIO 344, Exams of Molecular biology

Download Transcription and Translation - Lecture Notes | BIO 344 and more Exams Molecular biology in PDF only on Docsity! page 5.1 Revised 8/24/98 Transcription and Translation These terms describe the two steps used to transform the information carried in genes into useful products. The final product of some genes are RNA molecules. DNA Transcription RNA The final product for other genes are protein molecules. First the DNA gene is transcribed into a mRNA molecule and then the mRNA is translated into a protein. DNA Transcription mRNA Translation protein decoding the genetic code RNA Ribonucleic acid (RNA) is very much like DNA. It has a phosphodiester linked sugar backbone and uses primarily 4 different nitrogenous bases. The bases are A, G, C and U. U stands for uracil. It is like a different dialect of the same language. Two ways in which RNA is different than DNA. 1) The sugar is ribose NOT deoxyribose. 2) The nitrogenous base uracil is used in place of thymine. Uracil can also base pair with adenine. Uracil is a pyrimidine. Structure of RNA Some RNAs are essentially completely single stranded (mRNAs for instance). Some RNAs have some double stranded regions (rRNAs & tRNAs). IN RNA, G BASE PAIRS WITH C AND A BASE PAIRS WITH U (URACIL). What is RNA used for? The most well-known use of RNA is in transcription and translation. However, some viruses use RNA and not DNA as their genetic material. RIBOSE 1 OHOCH2 H H OH H OH H OH 23 4 5 OHOCH2 H H OH H OH H H 1 23 4 5 2-DEOXY-RIBOSE N H N O H CH3 THYMINE N H N O H URACIL RNA DNA Molecular Differences between Ribonucleic Acid (RNA) & 2-deoxy-ribonucleic acid (DNA). page 5.2 Revised 8/24/98 Before we continue some terminology Nucleotide Name Table Purines Pyrimidines Adenine (A) Guanine (G) Cytosine (C) Thymine (T) Uracil (U) Nucleotides in DNA deoxyadenylate deoxyguanylate deoxycytidylate deoxythymidylate or thymidylate Nucleotides in RNA adenylate guanylate cytidylate uridylate Abbreviations Nucleoside monophosphates AMP GMP CMP TMP UMP Nucleoside diphosphates ADP GDP CDP TDP UDP Nucleoside triphosphates ATP GTP CTP TTP UTP For deoxynucleotides add 'd' in front of the above three. Transcription Transcription: DNA is transcribed into RNA Transcripion The transcription machinery reads the nucleotides found in one of the DNA strands and builds a complementary RNA molecule. If it reads G in the DNA then it will put a C on the end of the RNA molecule. If it reads A in the DNA then it will add a U on the end of the RNA molecule. That is: C is complementary to G and U is complementary to A. C G U A GUAC GTAC CATG Free pool of ribonucleotides for use in making RNA A TT A C G G C A T Bases are being read from left to right A T first nucleotide to be read Double stranded DNA molecule Single stranded RNA molecule that is being built one nucleotide at a time. A 5' 3' 3' 5' 5' moving this way Notice: 1. In the diagram above it is the bottom DNA strand that is actually being read. 2. The RNA that is being synthesized is complementary and antiparallel to this DNA strand. Learn these two terms NOW!!! • Coding strand refers to the DNA strand that has the same sequence as the transcribed RNA. • Template strand refers the other strand. It is the template that was actually used to synthesize the RNA. It is antiparallel and complementary to the RNA. Antisense and sense strands are also antiparallel and complementary to one another. page 5.5 Revised 8/24/98 The genetic code is degenerate. In this context, degeneracy means that some amino acids are encoded by more than one codon. That is, some codons have synonyms. What the heck are stop codons? Translation is performed by the ribosomes. When a ribosome encounters a stop codon it dissociates from the mRNA and releases the protein. Stop codons are found at the end of the protein coding region. The codons UAA, UGA and UAG are stop codons. The genetic code is universal The genetic code shown above is used, essentially unchanged, in all organisms. There are minor exceptions to this truth, but you should think of these as different dialects of the same language. Be completely sure that you know how to read the genetic code table and could use it to translate a mRNA. The Process of Translation Translation is the process of reading the information carried in a mRNA molecule and using that information to direct the synthesis of a protein molecule. It is performed by the ribosomes using both mRNA, tRNA and amino acids. GUAC AA CG AA C GU A C C U A A A A A F S Y C L P H Q R I M T N K V A D E G W M T K A protein being synthesized. Proteins are polymers of amino acids. The letters FKSYCWLPHNRIMTKVADEG represent different amino acids. Amino acid pool. 20 different amino acids are used to synthesize proteins. Each letter represents a different amino acid. mRNA (made by the process of transcription) First codon to be read moving this way Tr ansl ati on 5' 3' Amino terminal end of the nascent polypeptide. The mRNA carries the information necessary to build a specific protein. Specifically, it describes the order of amino acids that the protein should contain. The mRNA is like a sentence. Each 'word' is 3 nucleotides long and is called a codon. There are no spaces and no punctuation. Each codon specifies a single amino acid. The translation machinery reads the mRNA a codon at time and joins together the amino acids specified by the mRNA. Proteins are synthesized in the amino terminus to carboxy terminus direction. In the example shown above: page 5.6 Revised 8/24/98 The first amino acid M is methionine it is encoded by the codon AUG. The second amino acid K is lysine it is encoded by the codon AAG. The third amino acid T is threonine it is encoded by the codon ACC. Not all RNAs are translated. In fact, only messenger RNA (mRNA) is translated. Both transfer RNA (tRNA) and the ribosomal RNAs (rRNA) are actually part of the translation machinery. What are protein molecules composed of? Proteins are polymers of amino acids. What are amino acids? These molecule are called amino acids because (with one exception) they each contain an amino group (NH2) and an acidic carboxylic group (COOH). General structure of amino acids----------> The alpha carbon is next to the COOH group. Attached to the α carbon is a hydrogen and a nitrogen. The nitrogen is part of an amino group. The side chain marked R is different in each amino acid and gives them their characteristic properties. αC COOH H H N2 R An exception to the above general structure. In the amino acid, proline, the nitrogen is not part of an amino groups but is part of an imino ring that involves the α carbon. The ring in proline causes it to be very rigid, and can produce a bend in a protein. None of the other R groups have rings that involve the α carbon. COOH C C CC HN H α imino ring Polypeptides or proteins are polymers of amino acids. The term polypeptide is usually reserved for very small proteins. In proteins, amino acids are joined to form polymers by the ribosomes. Condensation reaction: The amino acids are joined in a condensation reaction. This means that the reaction liberates a water molecule. The newly formed bond between the amino acids is called a peptide bond. O OαC H H N 3 R C O O αC H R C O + αC H R C O αC H R C O + H N 3 + H N3 + N H 1 2 1 2 Peptide Bond H2O page 5.7 Revised 8/24/98 Actually we should draw the peptide bond as ----------> Both O and N of the peptide bond have partial double bond characteristics. + -O C N H This double bond character causes the peptide bond to be flat (planar) and restricts the rotation about the peptide bond. Flexibility in proteins is primarily limited to the rotation around the α carbon's 2 single bonds. This is not true for proline. With proline the involvement of the α carbon in the imino ring further restriction the flexibility of the protein. You remember that nucleic acids have an inherent polarity and that they are synthesized in a particular direction. ------------------> Right? PPP P N1 P N2 P N3 N4 5' 3' OH PPP N5 5' 3' OH PPP P N1 P N2 P N3 P N4 5' 3' OH N5 Well, proteins also have an inherent polarity and are also synthesized in a particular direction. One end is called the amino terminus and the other the carboxy terminus. αC H R C O αC H R C O H N 3 + N H 1 2 αC H R C O N H O - 3 amino carboxy terminus terminus Proteins are synthesized in the amino to carboxy direction. αC H R C O αC H R C O H N3 + N H 1 2 O- αC H R C O αC H R C O H N3 + N H 1 2 αC H R C O N H O - 3 αC H R C O NH 3 O - 3 H2O page 5.10 Revised 8/24/98 Composition of Ribosomes 50 S subunit (large) 30 S subunit (small) 70 S subunit 5 S rRNA 120 bases + 23 S rRNA 2900 bases + 31 ribosomal proteins 16S rRNA 1540 bases + 21 ribosomal proteins + 60 S subunit (large) 40 S subunit (small) 80 S subunit + 5 S rRNA 120 bases + 5.8 S rRNA 160 bases + 28 S rRNA 4800 bases + 50 ribosomal proteins 18 S rRNA 1900 bases + 33 ribosomal proteins Prokaryotic ribosomes Eukaryotic ribosomes 5S 23 S 16 S 5S 5.8 S 28 S 18 S The ribosomal subunits are named according to their Svedberg coefficients (S). This is a unit of measure that describes the sedimentation rate of a particle in a centrifuge. It is particularly useful with very large macromolecular complexes. The greater the mass the larger is the Svedberg coefficient. Notice that the units are not additive. What do ribosomes contain? Ribosomes are composed to two different types of molecules: 1) RNA called rRNA or ribosomal RNA and 2) proteins, generically referred to as ribosomal proteins. In prokaryotes, the large ribosomal subunit contains two rRNAs (5S and 23S) and 31 different ribosomal proteins and the small subunit contains a single 16S rRNA and 21 different ribosomal proteins. Eukaryotic ribosomes are similar in composition to prokaryotic ribosomes. The exact size of the rRNAs and number of unique proteins differ between the two. One of the biggest differences is the presence of a third rRNA, the 5.8S rRNA, in the eukaryotic large subunit. Examine the diagram above very closely. Learn the sizes of the subunits and the rRNAs. page 5.11 Revised 8/24/98 rRNA has extensive secondary structure 5' 3' This diagram shows the secondary structure of the 16S rRNA from the bacteria, Escherichia coli. All rRNAs have extensive secondary and tertiary structure. The stems and loops are produced by the intramolecular base pairing between different parts of the molecule. This is a two- dimensional representation of the molecule. Actually, the molecule is folded in three dimensions to form a much more complex shape. This secondary structure is very similar, but not identical, in all organisms. This reflects the fact that the mechanisms of translation are evolutionarily strongly conserved. Function of the ribosomal components Translation requires both subunits. However, some of the labor is divided between the subunits. The small subunit recognizes the start codon and the large subunit synthesizes the peptide bond that joins the amino acids in a protein. Notice that the ribosomes are composed of both RNA and protein. Originally, it was thought that the rRNA served only a structural role, that it formed a scaffold used solely for the purpose of organizing the various ribosomal proteins. Furthermore, it was thought that it was the ribosomal proteins that did all of the catalytic work of protein synthesis. This is not entirely correct. It is true that the rRNA forms a scaffold, however, this is not its sole function. In the small subunit it is actually the 16S rRNA that recognizes the start codon of a mRNA. In the large subunit, it is the 23S rRNA that catalyzes peptide bond formation between amino acids. The various ribosomal proteins enhance both the specificity and efficiency of these processes. tRNA 2-dimensional model 3-dimensional model page 5.12 Revised 8/24/98 TΨC loop D loop Variable loop D stem Acceptor stem 3' acceptor end Anticodon stem Anticodon Amino acid D-loop D-Stem Acceptor Stem TΨC loop CCA end Anticodon loop 5' end Anticodon stem 3' amino acid acceptor site A tRNA is a single RNA chain that is folded into a two dimensional cloverleaf. This then folds in three dimensions to an L-like structure. tRNAs are small RNA molecules (usually in the range of 73 - 93 nucleotides) that participate in the translation of mRNAs. They are not part of the ribosome. But like rRNA they have extensive secondary structure produced by intramolecular base pairing between nucleotides. tRNAs are unique in that they contain many highly modified and unusual nucleotides. Immediately following transcription, the tRNA contains only the standard set of four nucleotides. The unusual nucleotides are produced by post-transcriptional enzymatic modifications. Some of the modifications help the cell to unambiguously recognize the tRNA. However, the function of most of the modifications is unknown. Take careful notice of the features common to all tRNAs. They are: XCCA, D loop, TΨC loop and Anticodon loop Notice that the tRNAs have 4 stems and 3 loops and that each of them has a name. Furthermore some tRNAs have a fourth loop called the Variable Loop. It is located between the TψC stem and the anticodon stem. The D loop is named for a modified nitrogenous base found within it; dihydrouracil. Similarly, the TψC loop, is named for four invariant nucleotides found within it. They are thymidylate, pseudouridylate, cytidylate, guanylate. The XCCA terminus is very important. XCCA refers to the nucleotides found at this position. X stand for any nucleotide, C for cytidylate and A for adenylate. The XCCA stem is the amino acid acceptor site. It is the tRNAs that actually decode the genetic code This is done via the anticodon loop. page 5.15 Revised 8/24/98 Here the tRNAarg is shown base pairing with the CGA codon for arginine. This is the usual type of base pairing with which you are familiar. 5' UCG Arginine 3' // GG GCU AUA UCA CAU AUA GGC AGC GUA UA // 5' mRNA Both of these codons encode the amino acid arginine (R). A tRNAarg is decoding the first arginine codon. Here we see the same tRNA base pairing with a different arginine codon. This the non-standard type of base pairing called Wobble Base Pairing. 5' UCG Arginine 3' // GG GCU AUA UCA CAU AUA GGC AGC GUA UA // 5' mRNA The same tRNA is decoding the second arginine codon! You can consider this wobble base pairing to be unique to the base pairing between the first position of the anticodon and the third position of the codon. The Genetic code table indicates that there are 6 different codons for arginine. They are CGU, CGC, CGA, CGG, AGA and AGG. Notice that these can be broken down into 2 groups that are identical in codon positions 1 and 2. Using wobble base pairing three different tRNAs can decode all 6 of these codons. page 5.16 Revised 8/24/98 These are the six CODONS that can encode arginine. 5' 3'CGC 1 2 3 5' 3'CGA 1 2 3 5' 3'CGG 1 2 3 5' 3'CGU 1 2 3 5' 3'AGA 1 2 3 5' 3'AGG 1 2 3 Each figure represents an arginine codon in a mRNA. It is written in the 5' to 3' direction. A minimum of three different tRNAs are required to decode all 6 of the above codons. They are: Each figure represents an anticodon in a tRNA. They are written in the 3' to 5' direction (backwards). 5'3' UCU 3 2 1 5'3' GCI 3 2 1 5'3' GCU 3 2 1 page 5.17 Revised 8/24/98 Below, I have spelled out how this happens. A minimum of two tRNAs are needed to recognize the CGX codon and a minimum of 1 tRNA is needed to recognize the AGA and AGG codons. wobble base pairing 5' 3'CGC 1 2 3 wobble base pairing 5'3' GCI 3 2 1 codon anticodon 5' 3'CGA 1 2 3 wobble base pairing 5'3' GCI 3 2 1 codon anticodon wobble base pairing 5' 3'CGU 1 2 3 5'3' GCI 3 2 1 codon anticodon 5'3' GCU 3 2 1 5' 3'CGG 1 2 3 codon anticodon 5' 3'CGA 1 2 3 normal base pairing 5'3' GCU 3 2 1 codon anticodon 5' 3'AGA 1 2 3 5'3' UCU 3 2 1 normal base pairing codon anticodon 5' 3'AGG 1 2 3 5'3' UCU 3 2 1 wobble base pairing codon anticodon Refer back to the Wobble Base Pairing Table until this makes sense.