1 00:00:00,008 --> 00:00:00,715 Hello. 2 00:00:00,715 --> 00:00:04,280 I'm Bob Leske of Johns Hopkins. 3 00:00:04,280 --> 00:00:07,550 We will examine the bacterial genome in this short lecture. 4 00:00:07,550 --> 00:00:12,862 Be sure to focus on the polycistronic nature of some transcripts. 5 00:00:12,862 --> 00:00:18,740 Unlike eukaryotes, most bacteria have a single chromosome. 6 00:00:18,740 --> 00:00:22,030 And in most but not all cases, that chromosome is circular. 7 00:00:24,590 --> 00:00:28,120 Polycistronic is an important term, meaning that there can be 8 00:00:28,120 --> 00:00:31,280 more than one protein coding reagent on an mRNA transcript. 9 00:00:34,950 --> 00:00:37,660 Examining that transcript more closely. 10 00:00:37,660 --> 00:00:42,190 You will find two untranslated regions that are not protein coated. 11 00:00:42,190 --> 00:00:43,670 Those come at either end of the sequence. 12 00:00:47,310 --> 00:00:52,020 We will look at the lactose operon, made famous by Jacob and Monod in the 1960s. 13 00:00:54,460 --> 00:00:57,200 And then to bring it back to bioinformatics, we will look to 14 00:00:57,200 --> 00:01:01,715 identify operons on an NCBI sequence record and on a gene prediction. 15 00:01:01,715 --> 00:01:08,780 [bbb] First, let's be aware that bacteria 16 00:01:08,780 --> 00:01:11,520 tend to have much smaller genomes than eukaryotes. 17 00:01:13,390 --> 00:01:16,460 Escherichia coli or what we call E. 18 00:01:16,460 --> 00:01:19,061 coli has a 4.6 megabase genome. 19 00:01:19,061 --> 00:01:20,860 That's 4.6 million base pairs. 20 00:01:25,070 --> 00:01:31,450 One of the simplest eukaryotes budding yeast has a genome of 12.1 megabases. 21 00:01:31,450 --> 00:01:33,470 Almost three times the size of E coli. 22 00:01:35,190 --> 00:01:37,610 And that's a very small eukaryotic genome. 23 00:01:40,670 --> 00:01:44,140 The human genome is 3.2 gigabases. 24 00:01:44,140 --> 00:01:46,140 That's 3.3 billion based pairs. 25 00:01:46,140 --> 00:01:48,920 And that's not even the largest genome. 26 00:01:48,920 --> 00:01:51,480 Some plants are even bigger in genome size than humans. 27 00:01:54,260 --> 00:01:57,063 Bacteria usually had a single circular chromosome. 28 00:01:57,063 --> 00:01:58,240 There are exceptions. 29 00:01:59,480 --> 00:02:03,540 Many also have extrachromosomal plasmids. 30 00:02:03,540 --> 00:02:06,660 Those are small, usually circular pieces of additional DNA. 31 00:02:11,160 --> 00:02:13,990 Here's a site map of the E coli genome. 32 00:02:13,990 --> 00:02:15,740 Look at the top where you see a zero. 33 00:02:15,740 --> 00:02:21,660 The 0 position represents what's called the origin of replication. 34 00:02:23,680 --> 00:02:26,166 Then the numbers increase clockwise and there 35 00:02:26,166 --> 00:02:28,530 are about 4 million 600 thousand or so. 36 00:02:28,530 --> 00:02:29,030 Now 37 00:02:32,560 --> 00:02:35,550 remember, there are still two strands of DNA. 38 00:02:35,550 --> 00:02:40,090 What we call the plus strand is arranged in this diagram, clockwise. 39 00:02:42,720 --> 00:02:44,850 However, there are genes on the minus or 40 00:02:44,850 --> 00:02:48,060 complimentary strand and they run in the other direction. 41 00:02:52,810 --> 00:02:54,780 So, I've mentioned polycistronic. 42 00:02:54,780 --> 00:02:57,720 In eukaryotes, the final splice mRNA 43 00:02:57,720 --> 00:02:59,900 usually only has one protein coding region. 44 00:03:01,440 --> 00:03:05,720 That means that only one protein is derived from the mRNA. 45 00:03:05,720 --> 00:03:08,250 Actually many copies of that same single protein. 46 00:03:11,430 --> 00:03:15,250 In bacteria, while mRNA splicing usually does not occur. 47 00:03:15,250 --> 00:03:17,840 There are often, but not always, more than 48 00:03:17,840 --> 00:03:20,390 one protein coding region in a prokaryotic mRNA. 49 00:03:23,320 --> 00:03:27,940 If say, an mRNA has five protein coding regions, then 50 00:03:27,940 --> 00:03:31,720 translation makes many molecules of each of those five proteins. 51 00:03:34,620 --> 00:03:36,250 And here's what it looks like. 52 00:03:36,250 --> 00:03:40,140 The diagram's a bit fuzzy but it displays the concept well. 53 00:03:40,140 --> 00:03:43,100 Prokaryote is at the top and the eukaryote is at the bottom. 54 00:03:44,820 --> 00:03:47,410 Prokaryotes tend to do it this way for efficiency. 55 00:03:52,660 --> 00:03:54,540 Here is a similar diagram. 56 00:03:54,540 --> 00:03:58,610 But you can see the untranslated regions or UTRs at either end. 57 00:04:01,830 --> 00:04:05,122 The one at the left of the mRNA is called the 5' UTR. 58 00:04:05,122 --> 00:04:08,880 The 5' refers to the chemistry of which 59 00:04:08,880 --> 00:04:11,369 carbon that phosphate attaches to on the sugar. 60 00:04:12,540 --> 00:04:14,440 Or to put it in another way, protein 61 00:04:14,440 --> 00:04:17,850 sequences go from N terminus to C terminus. 62 00:04:17,850 --> 00:04:24,702 DNA and RNA sequences go from 5' to 3', and both involve chemistry. 63 00:04:24,702 --> 00:04:30,590 The 5' UTR is everything to the left of the first AUG start codon. 64 00:04:33,670 --> 00:04:35,420 The 3' UTR is on the right. 65 00:04:37,820 --> 00:04:41,330 It's everything that is untranslated after the stop codon. 66 00:04:42,710 --> 00:04:46,644 Both UTRs are found in eukaryotes as well. 67 00:04:46,644 --> 00:04:51,780 So, from left to right on a bacterial mRNA, you have a 5' UTR. 68 00:04:53,110 --> 00:04:56,430 One or more codon regions then a 3' UTR. 69 00:05:00,420 --> 00:05:03,730 Here is the well-studied lactose operon. 70 00:05:03,730 --> 00:05:09,160 Bacteria prefer glucose as an energy source, but if there 71 00:05:09,160 --> 00:05:11,380 is no glucose to be found they'll deal with lactose. 72 00:05:12,700 --> 00:05:16,590 So they have to find away to turn on the genes involved in lactose synthesis. 73 00:05:18,110 --> 00:05:21,350 This describes some complex regulation and shows what's called the 74 00:05:21,350 --> 00:05:24,920 regulatory region of the gene, or in this case the operon. 75 00:05:26,240 --> 00:05:29,040 That's the term for the multiple genes on a single mRNA. 76 00:05:32,140 --> 00:05:34,732 Look primarily at the numbers below the first graphic. 77 00:05:34,732 --> 00:05:39,130 The +1 position is called the transcription start site. 78 00:05:40,190 --> 00:05:42,450 That is no the start codon. 79 00:05:42,450 --> 00:05:46,490 It is the start of the mRNA, transcription not translation. 80 00:05:48,070 --> 00:05:50,576 The mRNA is to the right of that +1 position. 81 00:05:51,790 --> 00:05:53,658 Or think of it this way. 82 00:05:53,658 --> 00:05:58,500 +1 refers to the beginning of the 5' UTR of the mRNA. 83 00:06:01,040 --> 00:06:04,060 The region to the left of the +1 is called the regulatory region. 84 00:06:05,592 --> 00:06:09,180 Here is where proteins bind that help control the level of transcription. 85 00:06:10,760 --> 00:06:14,170 A need for lactose digestion ultimately determines whether if 86 00:06:14,170 --> 00:06:17,010 the transcription of the lactose enzymes is on or off. 87 00:06:17,010 --> 00:06:24,120 The direction to the left, or to the 5' side, is frequently called upstream. 88 00:06:25,730 --> 00:06:30,719 The minus 35 position, is 35 bases upstream of the transcription start site. 89 00:06:33,260 --> 00:06:37,440 And to the right or to the 3' side is called downstream. 90 00:06:42,470 --> 00:06:47,460 In prokaryotes, you can have a single mRNA that contains more than one coding region. 91 00:06:47,460 --> 00:06:51,029 That would be very rare in eukaryotes, but very common in bacteria and archaea. 92 00:06:54,090 --> 00:06:58,410 This operon goes from position 70 to 6338. 93 00:06:58,410 --> 00:06:58,910 The 94 00:07:01,030 --> 00:07:04,820 mRNA would be larger since it would include the untranslated regions of the 95 00:07:04,820 --> 00:07:08,840 mRNA that come before the start codon and after the last dot codon. 96 00:07:11,050 --> 00:07:14,680 If you look at the annotation, you will see that this operon has four CDS regions. 97 00:07:14,680 --> 00:07:22,340 [bbb]. 98 00:07:22,340 --> 00:07:25,280 Here is an output for fgenesb@softberry.com. 99 00:07:25,280 --> 00:07:29,639 This is explained in a separate video on bacterial gene prediction. 100 00:07:31,740 --> 00:07:34,350 Note that there are four transcription units, meaning that 101 00:07:34,350 --> 00:07:37,220 four mRNAs come out of this region of genomic DNA. 102 00:07:38,960 --> 00:07:42,750 Of those four transcription unit, two are operons, which 103 00:07:42,750 --> 00:07:45,150 each have more than one CDS on that mRNA. 104 00:07:47,930 --> 00:07:49,150 The first mRNA. 105 00:07:50,350 --> 00:07:55,180 Has three CDS regions, Op 1, Op 2, and Op 3. 106 00:07:59,650 --> 00:08:04,520 Next are two single CDS transcription units, Tu 1and Tu 1. 107 00:08:05,760 --> 00:08:07,620 Why not Tu 2? 108 00:08:07,620 --> 00:08:11,670 That would imply a second CDS on an mRNA, that's how that numbering works. 109 00:08:14,280 --> 00:08:17,460 The last mRNA has 2 CDS regions, Op 1 and Op 2. 110 00:08:17,460 --> 00:08:20,880 Hopefully that explains the notation. 111 00:08:20,880 --> 00:08:21,380 To 112 00:08:27,560 --> 00:08:30,850 summarize, bacteria usually has circular genomes. 113 00:08:30,850 --> 00:08:33,620 Eukaryotes tend to have linear chromosomes and more than one. 114 00:08:36,340 --> 00:08:40,810 With prokaryotic genes, you don't have to deal with the splicing issue, usually. 115 00:08:42,620 --> 00:08:45,470 However, one complication is that multiple coding 116 00:08:45,470 --> 00:08:48,105 reagents often occur on a bacterial mRNA transcript. 117 00:08:50,910 --> 00:08:52,210 Finally, why do it this way? 118 00:08:53,320 --> 00:08:55,940 Bacteria waste little space in their genome. 119 00:08:55,940 --> 00:09:01,300 Efficiency allows for genes in a certain pathway to be transcribed onto one 120 00:09:01,300 --> 00:09:05,780 mRNA molecule so that all necessary enzymes can be translated into one shot. 121 00:09:07,540 --> 00:09:08,060 That's it for now.